Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF RISK ASSESSMENT OF PML AND RELATED APPARATUS
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] The present application claims the benefit of and the priority to an
application for "Risk Stratification of Patients Receiving VLA-4 Blocking
Agents" filed
on 17 October 2011 with the European Patent Office, and there duly assigned
serial
number EP 11 185 439. The present application further claims the benefit of
and the
priority to an application for "Methods of Risk Assessment of PML" filed on 07
March
2012 with the European Patent Office, and there duly assigned serial number EP
12 158
369. The contents of said applications filed on 17 October 2011 and 07 March
2012 are
incorporated herein by reference for all purposes, including an incorporation
of any
element or part of the description, claims or drawings not contained herein
and referred
to in Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.
FIELD OF THE INVENTION
[002] The present invention generally relates to evaluating the immune
response of a subject to a therapeutic agent. The present invention also
relates to a
method of treating a subject with a demyelinating disease or an autoimmune
disease. In
another aspect, the present invention concerns the identification of patients
at lower or
higher risk for developing progressive multifocal leukoencephalopathy. More
specifically, the present invention relates to a method of assessing the risk
of occurrence
of progressive multifocal leukoencephalopathy (PML) in a subject. The
invention also
relates to a method of stratifying a subject undergoing VLA-4 blocking agent
treatment
for suspension of this VLA-4 blocking agent treatment. Provided is further a
method of
administering a VLA-4 blocking agent to a subject so as to avoid the
additional
occurrence of PML.
BACKGROUND OF THE INVENTION
[003] Multifocal Progressive multifocal leukoencephalopathy (PML) is a
severe, rapidly progressive disease that destroys the myelin coating which
protects nerve
cells. It is caused by the JC virus, a common polymavirus. Through poorly
understood
interactions between host and viral factors, JCV undergoes alterations in the
regulatory
region and mutations in the coat protein VP1 to cause lytic infection of
myelin-producing
oligodendrocytes, leading to development of progressive multifocal
leukoencephalopathy. PML occurs generally in severely immunocompromised
individuals and in patients receiving certain immunosuppressive
therapiestherapy of
multiple sclerosis and Crohn's disease.
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[004] Multiple sclerosis (MS) is a chronic, inflammatory central nervous
system (CNS) disease characterized pathologically by demyelination. MS has
also been
classified as an autoimmune disease. MS disease activity can be monitored by
cranial
scans, including magnetic resonance imaging (MRI) of the brain, accumulation
of
disability, as well as rate and severity of relapses. There are five distinct
disease stages
and/or types of MS, namely, (1) benign multiple sclerosis; (2) relapsing-
remitting
multiple sclerosis (RRMS); (3) secondary progressive multiple sclerosis
(SPMS); (4)
progressive relapsing multiple sclerosis (PRMS); and (5) primary progressive
multiple
sclerosis (PPMS).
[005] The VLA-4 blocking agent, Natalizumab, was first approved in 2004 by
the U.S. Food and Drug Administration (FDA) for the treatment of multiple
sclerosis. It
was subsequently withdrawn from the market after it was linked with three
cases of
PML. After a review of safety information and no further deaths, the drug was
returned
to the US market in 2006 under a special prescription program.
[006] So far there have been over 150 cases of JCV-induced progressive
multifocal leukencephalopathy (PML) associated with the treatment of MS
patients with
Natalizumab. It is still largely unknown how the treatment with blocking VLA-4
interferes with JC virus control or immune surveillance. Crohn's disease is a
type of
inflammatory bowel disease. It typically manifests in the gastrointestinal
tract and can be
categorized by the specific tract region affected. It is thought to be an
autoimmune
disease, in which the body's immune system attacks the gastrointestinal tract,
causing
inflammation of gastrointestinal tract. The disease manifestations usually are
isolated to
the digestive tract, but other manifestations such as inflammation of skin
structures, the
eyes, and the joints have been well described. The disease is known to have
spontaneous
exacerbations and remissions. Unfortunately, the cause of Crohn's disease is
not known,
and there is no known cure for Crohn's disease.
[007] Crohn's disease has an immune response pattern that includes an
increased production of interleukin-12, tumour necrosis factor (TNF) and
interferon-y.
Tumor necrosis factor (TNF) has been identified as an important cytokine in
the
pathogenesis of Crohn's disease, with elevated concentrations playing a role
in
pathologic inflammation. The increased production of TNF by macrophages in
patients
with Crohn's disease results in elevated concentrations of TNF in the stool,
blood, and
mucosa. In recent years, biologic response modifiers that inhibit TNF activity
have
become potential therapies for treating Crohn's disease.
[008] The humanized monoclonal immunoglobulin Natalizumab is used in the
treatment of both MS and Crohn's disease. Natalizumab is both clinically
effective and
generally well-tolerated. However, Natalizumab treatment for longer than 18
months has
been found to be associated with an enhanced risk of developing PML. PML has
almost
exclusively been found in immunocompromised individuals, especially in
subjects with
reduced cellular immunity. It has also been reported in in rheumatic diseases.
PML has
for example been found in individuals with hematological malignancies and
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lymphoproliferative diseases, individuals with Hodgkin's lymphoma, individuals
with
systemic lupus erythematosus or subjects receiving immunosuppressive
medication such
as transplant patients. In addition to Natalizumab therapy, PML has also been
found to be
associated with therapy using the monoclonal antibodies Rituximab, used in the
treatment of lymphomas, leukemias, transplant rejection and certain autoimmune
disorders, and Efalizumab, formerly used in the treatment of autoimmune
diseases, in
particular psoriasis. In view of the risk of PML, Efalizumab has currently
been
withdrawn from the U.S. market. Natalizumab was now restricted as a
monotherapy for
relapsing remitting multiple sclerosis (RRMS) patients with high disease
activity.
[009] So far there have been over 150 cases of JCV-induced PML associated
with the treatment of MS patients with Natalizumab, where the mortality rate
of so far
20%. It is still largely unknown how the treatment with blocking integrin
a4131NLA-4
interferes with JCV control or immune surveillance (Tan, C.S, and Koralnik,
I.J., Lancet
Neurol. (2010) 9, 4, 425-437).
[010] Prognosis of PML is poor, since no specific therapy is available. While
only 20% of the Natalizumab-treated PML patients so far died, the overall
mortality of
PML has been reported to be above 50%. In the absence of any therapy it would
be
particularly helpful to be able to predict the risk whether an individual is
likely to
develop PML. Hence, there exists a need for means to determine at an early
stage, i.e.
before the onset of the disease, whether an HIV positive individual is likely
to suffer
from PML.
[011] Recent studies suggest that patients under treatment with the very late
activation antigen 4 (VLA-4, integrin a4131) blocking agent Natalizumab for
more than
12 months are at elevated risk for PML, with the risk increasing after
approximately 18
months of treatment, and can reach risk levels of up to 1:120. It is not known
if the risk
of developing PML continues to increase, remains the same, or decreases after
a patient
has been on Natalizumab for more than three years. Since there is a clear risk
association
between Natalizumab and the development of PML after long-term treatment of
the
VLA-4 blocking agent Natalizumab, there is an urgent need to identify those
patients
who are more prone to PML. However, only few candidates have evolved: (1)
treatment
duration, (2) pre-treatment with immunosuppressive drugs, and (3) presence of
JCV
antibodies in serum.
[012] U.S. Pat. Pub. No. 2010/0196318 discloses testing for serum anti-JCV
antibody prior to initiating Natalizumab therapy in patients. However, the
detection of
JCV antibody in an individual does not predict the risk for PML and therefore
cannot
advise a medical professional whether or not to continue the treatment. U.S.
Pat. Pub.
No. 2009/0216107 discloses a method of screening patients undergoing
Natalizumab
treatment by testing the patient's cerebrospinal fluid to detect the presence
of
cytomegalovirus, JCV, Toxoplasma gondii, Epstein-Barr virus, Cryptococcus
neoformans and tuberculosis by PCR, as well as examining the retinal status to
detect the
presence of ocular cytomegalovirus. If an indication of the presence of the
virus is
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detected, Natalizumab treatment should be discontinued. However, such methods
are
only precautionary measures which also do not indicate a risk of developing
PML. There
still remains a need to develop a method to determine the susceptibility to
PML for
patients who receive VLA-4 blocking agent on an individual basis. It would be
advantageous if the determination could help the practitioner to identify
patients who are
particularly prone to PML or stop the treatment in time before the immune
competence
of the subject deteriorates.
[013] It is therefore an object of the present invention to provide a method
and
apparatus that is suitable for determining the risk for PML development in a
subject. It
would be advantageous if such method and apparatus can be used to monitor the
immune
competence of patients receiving or expected to receive Natalizumab thus to
avoid the
possible development of PML or even another complication at a later stage. It
is a further
object of the invention to provide a method for assessing the likelihood of
PML
occurrence in a subject suffering from HIV. These objects are solved by the
method of
claim 1. Also provided herein are apparatuses such as kits and assays related
to these
purposes.
[014] It is also another objective of the present invention to develop an
improved method of treating patients with VLA-4 blocking agents.
SUMMARY OF THE INVENTION
[015] The present invention generally relates to the determination of a
subject's
immune competence. More specifically, the present invention provides inter
alia a
method for assessing the susceptibility to a condition associated with JC
virus and a
method of PML risk stratification, and means for the methods. In one aspect,
this
disclosure provides a method for determining the susceptibility of a patient
undergoing
VLA-4 blocking agent treatment to a JCV-induced disease.
[016] The present invention is based, at least in part, on the finding that
the
binding of VLA-4 influences the expression of adhesion molecules CD62L and LFA-
1
and basic immune cell functions such as migratory capacity. Without being
limited to
any particular theory, it has been discovered that some adhesion molecules,
CD62L and
LFA-1 included, are differentially expressed in patients who developed PML.
[017] The methods and uses provided by the present invention are based on
employing L-selectin (CD62L) and optionally lymphocyte function-associated
antigen-1
(LFA-1) as a biomarker for identifying a predisposition of a subject of
developing PML.
Use of such molecules as biomarkers, in conjunction with further biomarkers or
tests,
provides indication of which patients are more likely to suffer from PML. The
biomarkers provided in the present invention can assist physicians in the
determination
of appropriate therapeutic regimen. In the context of the present invention
CD62L levels
may be determined using any desired technique. In some embodiments means may
be
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employed that indirectly indicate CD62L levels and optionally LFA-1 levels,
for
example by assessing indicators from which levels of CD62L and optionally LFA-
1 can
be inferred.
[018] Accordingly, the biomarkers provided in the present invention may be
advantageously used to assess the immune competence of subjects, preferably
subjects
who are receiving or expected to receive long-term VLA-4 blocking agent
treatment, as
well as to determine the risk of the subject to suffer from PML.
[019] According to a first aspect, the present invention provides a method of
assessing the risk of occurrence of PML in a subject. The method can be seen
as method
of treating patients with a demyelinating disease or an autoimmune disease,
including,
but not limited to, multiple sclerosis (MS), Crohn's disease (CD) and
rheumatoid
arthritis. As mentioned earlier, a number of MS and CD patients receiving VLA-
4
blocking agents may become susceptible to PML. Therefore, in another aspect
the
present invention provides a method of treatment comprising administering VLA-
4
blocking agents, measuring the expression of CD62L and optionally LFA-1 on T
cells in
sample from a patient to be treated, and stopping or continuing the
administration of
VLA-4 blocking agents based on the measured level of the expression.
[020] In another aspect, the present invention provides a way of screening
such
patients who are more prone to PML. The method generally includes providing a
sample
from the subject and detecting the level of CD62L expressing T cells in the
sample from
the subject.
[021] In another aspect, the present invention provides a method of predicting
whether a patient is at risk of developing PML. The method comprises a)
measuring the
expression of at least one biomarker from T cell, and b) comparing the
expression with a
reference value. The term "predict" generally means to determine in advance.
As used
herein, the term "predict" means to determine the risk for PML or assess the
risk or
occurrence for PML if the patient continues to receive VLA-4 blocking agent
treatment.
The present invention is also related to a kit comprising CD62L binding assay
and
optionally LFA-1 binding assay and use thereof for determining the
susceptibility of a
patient to progressive multifocal leukoencephalopathy. According to a
particular
embodiment of the present invention, the T cells are CD3 ' T cells. In one
embodiment
the method includes detecting the level of CD62L expressing T cells in a
sample from
the subject. According to a particular embodiment the method according to the
first
aspect further includes detecting the level of LFA-1 expressing T cells in the
sample
from the subject. According to some embodiments of the method according to the
first
aspect, the expression of CD62L is monitored at certain, e.g. predetermined,
time
intervals. According to a particular embodiment of the method according to the
first
aspect, the expression of both CD62L and LFA-1 is monitored at certain, e.g.
predetermined, time intervals.
[022] According to a further embodiment of the method according to the first
aspect, the subject is diagnosed as being in need of treatment with a VLA-4
blocking
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agent. In such an embodiment the level of CD62L expressing T cells and
optionally
LFA-1 expressing T cells in the sample from the subject may be analysed.
[023] In one embodiment of the method according to the first aspect a
decreased level of CD62L expressing T cells and optionally of LFA-1 expressing
T cells,
relative to a threshold value, indicates a risk of occurrence of PML. A level
of CD62L
expressing T cells and optionally of LFA-1 expressing T cells that is about at
a threshold
value or above a threshold value indicates no risk of occurrence of PML when
compared
to healthy subjects.
[024] According to an embodiment of the method according to the first, the
second, the third and/or the fourth aspect, the method includes comparing the
level of
CD62L and optionally LFA-1 expressing T cells in the sample to a threshold
value.
[025] According to further embodiments of the method according to the first,
the second, the third and/or the fourth aspect, the method includes
determining the
migration of CD45 'CD49d immune cells, such as CD45 'CD49d' T cells. In some
embodiments migration is measured using a transendothelial chemotaxis assay.
In some
embodiments migration is measured using a chemotaxis assay, for instance
employing a
blank permeable membrane.
[026] In a related second aspect the invention provides a method of screening
one or more individuals for susceptibility to a condition associated with JCV
infection.
The method generally includes providing a sample from each of the one or more
subjects. The method includes detecting the level of CD62L expressing T cells
and/or
detecting the level of LFA-1 expressing T cells in the sample from each of the
one or
more subjects.
[027] In some embodiments of the method according to the second aspect an
altered, such as a decreased or an increased, level of CD62L and/or LFA-1
expressing T
cells, relative to a threshold value, may indicate a susceptibility to a
condition associated
with JCV infection. In such embodiments a method according to the second
aspect may
include determining that the subject is susceptible to a condition associated
with JCV
infection.
[028] In one embodiment of the method according to the second aspect, a
decreased level of CD62L expressing T cells and/or of LFA-1 expressing T
cells, relative
to a threshold value, indicates that the subject is susceptible to a condition
associated
with JCV infection. A level of CD62L expressing T cells and optionally of LFA-
1
expressing T cells that is about at a threshold value or above a threshold
value indicates
that the subject is not susceptible to a condition associated with JCV
infection when
compared to healthy subjects.
[029] According to a particular embodiment, the method according to the
second aspect includes comparing the level of CD62L and optionally LFA-1
expressing
T cells in the sample to a threshold value.
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[030] In a further aspect there are provided methods of monitoring the
susceptibility of a JCV related condition in a subject. The methods include
monitoring
the level of CD62L1 expressing T cells of the subject. Generally these T cells
are
included, including provided, in a sample from the subject. In some
embodiments the
method further includes monitoring the level of LFA-1 expressing T cells of
the subject.
Monitoring the expression of CD62L and optionally LFA-1 on T cells is
generally
carried out using a sample from the subject. Monitoring may be carried out at
predetermined time intervals. In some embodiments monitoring begins prior to a
treatment. In some embodiments a respective treatment may be a VLA-4 blocking
agent
treatment.
[031] In a related aspect there is provided a method of monitoring the
susceptibility of a subject to PML. The method includes monitoring the level
of
expression of CD62L on T cells. In some embodiments the method further
includes
monitoring the level of expression of LFA-1 on T cells. Generally these T
cells are
included, including provided, in a sample from the subject. In a further
aspect there is
disclosed a method of screening patients who are known or suspected to be
prone to
occurrence of PML. The method generally includes detecting the level of CD62L
expressing T cells in a sample from the subject. In some embodiments the
method further
includes detecting the level of LFA-1 expressing T cells in the sample. The
method may
also include comparing the result to a threshold value.
[032] According to a third aspect, the invention provides a method of
stratifying a subject that/who is undergoing VLA-4 blocking agent treatment
for
suspension of VLA-4 blocking agent treatment. The method generally includes
providing
a sample from the subject. The method further includes detecting the level of
T cells in
the sample from the subject, with the T cells expressing CD62L. In some
embodiments
of the method according to the third aspect, the T cells are expressing both
CD62L and
LFA-1.
[033] In some embodiments of the method, an altered, such as a decreased or
an increased, level of CD62L and optionally LFA-1 expressing T cells, relative
to a
threshold value, may indicate a risk of occurrence of PML. In such embodiments
a
method according to the third aspect may include stratifying the subject for
suspension of
VLA-4 blocking agent treatment.
[034] In one embodiment of the method, a decreased level of CD62L
expressing T cells and optionally of LFA-1 expressing T cells expressing T
cells, relative
to a threshold value, indicates a risk of occurrence of PML. A level of CD62L
expressing
T cells and optionally of LFA-1 expressing T cells that is about at a
threshold value or
above a threshold value indicates no risk of occurrence of PML when compared
to
healthy subjects.
[035] According to a particular embodiment, the method includes comparing
the level of CD62L and optionally LFA-1 expressing T cells in the sample to a
threshold
value.
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[036] According to a fourth aspect, the invention provides a method of
stratifying a subject in need of VLA-4 blocking agent treatment for risk of
PML
occurrence. The method generally includes providing a sample from the subject.
The
method further includes detecting the level of T cells which express CD62L, in
the
sample from the subject.
[037] In some embodiments of the method according to the fourth aspect an
altered, such as a decreased or an increased, level of CD62L expressing T
cells, relative
to a threshold value, may indicate an increased risk of PML occurrence. In
such
embodiments a method according to the fourth aspect may include stratifying
the subject
for risk of PML occurrence. According to a particular embodiment, the method
according to the fourth aspect includes comparing the level of CD62L
expressing T cells
in the sample to a threshold value.
[038] According to a further aspect, the invention relates to the in-vitro use
of a
capture probe, which is specific for CD62L, for assessing the risk of
occurrence of PML
in a subject.
[039] In typical embodiments the capture probe is an immunoglobulin or a
proteinaceous binding molecule with immunoglobulin-like functions, with the
binding
molecule or the immunoglobulin being specific for CD62L.
[040] The subject may in some embodiments suffer from an autoimmune
disease, such as HIV, The subject may be undergoing treatment with one or more
VLA-4
blocking agents.
[041] According to a further aspect the invention relates to the in-vitro use
of a
capture probe, which is specific for LFA-1, for assessing the risk of
occurrence of PML
in a subject. The subject may in some embodiments suffer from an autoimmune
disease.
In some embodiments the subject may undergo treatment with one or more VLA-4
blocking agents. In typical embodiments the capture probe is an immunoglobulin
or a
proteinaceous binding molecule with immunoglobulin-like functions specific for
LFA-1.
[042] According to another aspect, the invention relates to the in-vitro use
of a
capture probe, which is specific for at least one of CD11A, CD18 and LFA-1,
for
assessing the risk of occurrence of PML in a subject.
[043] In typical embodiments the capture probe is an immunoglobulin or a
proteinaceous binding molecule with immunoglobulin-like functions specific for
at least
one of CD11A, CD18 and LFA-1.
[044] According to a further aspect, the invention relates to the in-vitro use
of a
capture probe, which is specific for at least one of CD11A, CD18 and LFA-1,
for
stratifying a subject undergoing VLA-4 blocking agent treatment for suspension
of the
VLA-4 blocking agent treatment.
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[045] In typical embodiments the capture probe is an immunoglobulin or a
proteinaceous binding molecule with immunoglobulin-like functions specific for
at least
one of CD11A, CD18 and LFA-1.
[046] According to another aspect the invention relates to the use of a CD62L
and optionally LFA-1 binding assay kit for determining the susceptibility of a
subject
undergoing or intended to undergo VLA-4 blocking agent treatment to PML.
[047] According to a particular embodiment of the use according to this aspect
the CD62L and optionally LFA-1 binding assay kit employs a CD62L capture probe
and
optionally a LFA-1 capture probe. According to a further particular embodiment
of the
use, the subject is a multiple sclerosis or a Crohn's disease patient.
[048] According to a further aspect the invention relates to the use of a
CD62L
binding assay kit for determining the susceptibility of an HIV positive
subject to PML.
The CD62L binding assay kit comprises a CD62L capture probe.
[049] According to another aspect there is provided a method of treating a
subject. The method includes administering one or more VLA-4 blocking agents
to the
subject. The method may further include detecting the level of expression of
one or more
biomarkers disclosed herein on T cells, such as CD3-' T cells, of the subject.
In some
embodiments the expression of the one or more biomarkers on T cells is
monitored.
Generally these T cells are included, including provided, in a sample from the
subject.
The method may also include determining migration of immune cells, such as
CD45-tD49-' cells and T cells. In some embodiments, a respective biomarker is
one or
more of CD62L and LFA-1. In some embodiments the method may include both (i)
detecting the level of expression of the one or more biomarkers on T cells and
(ii)
determining migration of immune cells. In some embodiments the subject may be
suffering from a demyelinating or autoimmune disorder.
[050] The subject is in some embodiments suffering from a pathologic
inflammatory disease within the CNS. The subject may in some embodiments be
diagnosed to have an autoimmune disease, such as multiple sclerosis, e.g.
relapsing-
remitting multiple sclerosis and secondary progressive multiple sclerosis or
Crohn's
disease. In some embodiments the VLA-blocking agent is CD49d specific, i.e.
specific
for the integrin ct4 chain. Examples of a suitable VLA-4 blocking agent
include, but are
not limited to, the monoclonal antibodies Natalizumab, HP2/1, HP1/3, HP1/2,
including
humanized HP1/2, HP1/7, HP2/4, B-5G10, TS2/16, L25, P4C2, AJM300 and the
recombinant anti-VLA4 antibodies described in U.S. patents US 6,602,503 and US
7,829,092, a low molecular weight VLA-4 antagonist such as SB-683699, a CS-1
peptidomimetic as disclosed in e.g. U.S. patents US 5,821,231, US 5,869,448,
US
5,869,448, US 5,936,065, US 6,265,572, US 6,288,267, US 6,365,619, US
6,423,728,
US 6,426,348, US 6,458,844, US 6,479,666, US 6,482,849, US 6,596,752, US
6,667,331, US 6,668,527, US 6,685,617, US 6,903,128 or US 7,015,216, a
phenylalanine
derivative as disclosed in e.g. U.S. patents US 6,197,794, US 6,229,011, US
6,329,372,
US 6,388,084, US 6,348,463, US 6,362,204, US 6,380,387, US 6,445,550, US
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6,806,365, US 6,835,738, US 6,855,706, US 6,872,719, US 6,878,718, US
6,911,451,
US 6,916,933, US 7,105,520, US 7,153,963, US 7,160,874, US 7,193,108, US
7,250,516
or US 7,291,645, alphafeto protein, a beta-amino acid compound as disclosed in
e.g.
patent applications US 2004/0229859 or US 2006/ 0211630, a semi-peptide
compound
as disclosed in e.g. patent 6,376,538, the tripeptide Leu-Asp-Val and a
pegylated
molecule as disclosed in U.S. patent application US 2007/066533 or U.S. patent
US
6,235,711.
[051] In another aspect there is provided a method of treating an autoimmune
disease in a subject so as to avoid the additional occurrence of PML. The
method
includes administering one or more VLA-4 blocking agents to the subject,
generally an
effective amount of the VLA-4 blocking agent(s), over a period of time,
followed by
discontinuing the administration for a period of time.
[052] In relation to the method of treatment, the present invention provides
the
following items:
Item 1. A method of treating a patient comprising administering to the patient
VLA-4
blocking agent, detecting the level of T cells expressing L-selectin (CD62L)
in a
sample from the patient, and continuing or stopping the administration based
on the
level of expression, wherein the administration is stopped if a decreased
level of
CD62L expressing T cells, relative to a threshold value, is detected.
Item 2. The method of item 1, wherein the patient suffers from a demyelinating
disease
or an autoimmune disease.
Item 3. The method of item 2, wherein the VLA-4 blocking agent is an
immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like
functions.
Item 4. The method of any one of items 1 to 3, further comprising detecting
the level of
T cells expressing lymphocyte function-associated antigen-1 (LFA-1) in the
sample,
wherein the administration is stopped if a decreased level of LFA-1 expressing
T cells,
relative to a threshold value, is detected..
Item 5. The method of any one of items 1 to 4, further comprising further
administering to the patient VLA-4 blocking agent the treatment if an
increased level of
CD62L, relative to the threshold value, is detected after the treatment is
stopped.
Item 6. The method according to any one of the preceding items, wherein the
method
further comprises determining the migration of CD45 'CD49d immune cells.
Item 7. The method according to item 6, wherein the immune cells are T cells.
Item 8. The method of any one of items 1 to 7, wherein the sample is one of a
blood
sample, a blood cell sample, a lymph sample and a sample of cerebrospinal
fluid.
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Item 9. The method of any one of items 1 to 8, wherein detecting the level of
CD62L
expressing T cells comprises detecting at least one of:
(i) the number of T cells in the sample from the subject that have CD62L on
the
cell surface,
(ii) the amount of CD62L present on T cells of the sample from the subject,
and
(iii) the amount of nucleic acid formation from the SELL gene encoding
CD62Lin T cells of the sample from the subject.
Item 10. The method of any one of items 4 to 9, wherein detecting the
level of
LFA-1 expressing T cells comprises detecting at least one of:
(i) the number of T cells in the sample from the subject that have LFA-1 on
the
cell surface,
(ii) the amount of LFA-1 present on T cells of the sample from the subject,
and
(iii) the amount of nucleic acid formation from the ITGAL gene encoding
CD11A and the ITGB2 gene encoding CD18 in T cells of the sample from the
subject.
Item 11.The method of any one of items 1 to 10, wherein the T cells are at
least one of
CD4 ' T cells and CD8 T cells.
Item 12.The method of any one of items 1 to 11, comprising repeatedly
detecting the
level of at least one of CD62L expressing T cells and LFA-1 expressing T cells
in a
sample from the subject.
Item 13.The method of any one of items 11 to 12, wherein (i) detecting the
number of T
cells in the sample that have CD62Lon the cell surface and/or (ii) detecting
the amount
of CD62L present on T cells of the sample comprises contacting T cells in/of
the
sample with a capture probe, the capture probe being specific for CD62L, and
detecting
the amount of the capture probe binding to CD62L.
Item 14.The method of any one of items 11 to 16, wherein (i) detecting the
number of T
cells in the sample that have LFA-1 on the cell surface and/or (ii) detecting
the amount
of LFA-1 present on T cells of the sample comprises contacting T cells in/of
the sample
with a capture probe, the capture probe being specific for LFA-1, and
detecting the
amount of the capture probe binding to LFA-1.
[053] In some embodiments the expression of the one or more biomarkers on T
cells, including CD4 ', CD3 ' or CD8' T cells are monitored in a sample from
the subject.
In some embodiments, a respective biomarker is one or more of CD62L and LFA-1.
In
some embodiments discontinuing the administration of the one or more VLA-4
blocking
agents is effected after detection of a decreased or an increased level of the
one or more
biomarkers on T cells, including CD4 ', CD3 ' or CD8' T cells, relative to a
threshold
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value. For example, the level of CD62L expressing T cells in the subject and
optionally
the level of LFA-1 expressing T cells in the subject may be altered.
[054] Determining the level of CD62L and optionally LFA-1 expressing T cells
in any of the above aspects and embodiments may include detecting the number,
percentage or level of T cells in the sample from the subject that have CD62L
and
optionally LFA-1 on the cell surface. Determining the level of CD62L and
optionally
LFA-1 expressing T cells may also include detecting the amount or level of
CD62L and
optionally LFA-1 present on T cells of the sample from the subject.
Determining the
level of CD62L expressing T cells may also include detecting, in T cells of
the sample
from the subject, the amount or level of nucleic acid formation from the SELL
gene
encoding CD62L. Determining the level of LFA-1 expressing T cells may also
include
detecting the amount or level of nucleic acid formation from the ITGAL gene
encoding
CD11A and/or the ITGB2 gene encoding CD18.
BRIEF DESCRIPTION OF THE DRAWINGS
[055] The invention will be better understood with reference to the detailed
description when considered in conjunction with the non-limiting examples and
the
accompanying drawings.
[056] Figure 'IA depicts the percentage (%) of CD62L surface expressing
CD3 ' CD4 ' T cells, as determined by flow cytometric measurements using
peripheral
blood derived mononuclear cells (PBMC). Cells were isolated from EDTA blood by
density gradient centrifugation, frozen, thawed for analysis, and stained with
fluorescence labelled immunoglobulins against CD3, CD4 and CD62L. Cells were
gated
as shown in Figure 1C. The boxes in Fig. lA represent 50% of each cohort (25th-
75th
percentile) while 80% of all individuals reside within the limits of each box
and its
whiskers (10t1-90t1 percentile). The line within the boxes indicates the mean,
the plus (+)
represents the median of the respective cohort. Each dot represents an
individual patient.
The white box represents 21 control subjects without any acute or chronic
disorder
(healthy controls). The dotted box represents subjects diagnosed for MS, who
are in
stable condition and did not receive any prior immune-modulating treatment (MS
naïve).
The light grey box represents patients diagnosed for MS, who received baseline
treatments other than Natalizumab as lined-out in Fig. 15. These blood
withdrawings
took place right before the escalation to Natalizumab therapy (MS baseline).
The dark
grey box indicates patients diagnosed for MS, who after receiving baseline
treatments as
lined-out in Fig. 15 received Natalizumab continuously for 18 months or longer
(18-66
months of Natalizumab treatment, MS Natalizumab). The six numbered MS
(Natalizumab) pre-PML patients all match the criteria of the dark grey cohort,
but
developed PML later on at different time points throughout Natalizumab long-
term
therapy as lined out in Fig. 15. The dotted line indicates the threshold for
increased PML
risk under long-term Natalizumab therapy (mean of the dark grey cohort minus
two times
its standard deviation).
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[057] Figure 1B depicts the percentage (%) of CD62L surface expressing
CD3 ' CD4 T cells. See the explanation for Fig. lA for details. The MS
(Natalizumab)
acute- and post-PML cohorts both match the dark grey cohort but were sampled
after
PML onset, either while suffering from acute PML (MS (Natalizumab) acute-PML)
or
after PML subsided (MS (Natalizumab) post-PML, e.g. the beginning of immune
reconstitution inflammatory syndrome (IRIS). Two patients with other
monoclonal
antibody-associated PMLs, one suffering from severe psoriasis treated with
Efalizumab
and one suffering from B-cell lymphoma treated with Rituximab (other
monoclonal
antibody-associated acute-PML), and seven HIV/AIDS PML patients (HIV-
associated
acute-PML) served as additional PML controls. The dashed lines indicate
sequential
samples, if identical patients were available at different time points during
disease
development.
[058] Figure 1C shows illustrative flow cytometry measurements with gating
to life lymphocytes, CD3 ' T cells as well as CD4+ and CD8+ T cells.
[059] Figure 1D depicts data of flow cytometric measurements of peripheral
blood derived mononuclear cells (PBMC). Cells were isolated from EDTA blood
(EDTA: 1.2 to 2 mg/ml blood) by density gradient centrifugation and
subsequently
frozen. For analysis, cells were thawed and stained with fluorescence labelled
antibodies
against CD3, CD4 and CD62L. 200,000 cells were used per staining. After flow
cytometry measurement, cells were first gated to life lymphocytes, then CD3 '
cells then
CD4' cells and finally on CD62L' cells (cf. also Fig. 1C). The graph depicts
CD3 ' CD4'
living lymphocytes that are positive for CD62L (1-selectin) expression. The
groups are as
following: HD = healthy controls without any pathology or treatment; NAT =
patients
suffering from relapsing/ remitting multiple sclerosis long-term treated with
Natalizumab
(18+ months of treatment); HIV = patients suffering from HIV infection treated
with
HAART medication; HIV PML = patients suffering from HIV infection treated with
HAART medication that developed PML alongside therapy.
[060] Figure 2 shows percentages of CD14 ' monocytes, CD4 ' and CD8' T
cells, CD19' B cells, and CD56' NK cells (of PBMC) in peripheral blood of
patients
receiving long-term Natalizumab therapy (>18 months). White dots represent
healthy
donors (n=16-39), black dots represent untreated MS patients (n=12), and grey
dots
represent Natalizumab patients treated >18 months continuously (n=34).
Significance of
differences between the groups is indicated by asterisks (*p<0.05, **p<0.01,
***p<0.001).
[061] Figure 3 depicts data analysis of flow cytometric measurements of
peripheral blood derived mononuclear cells (PBMC). EDTA blood was obtained
from
patients and healthy control subjects as indicated above, PBMC were isolated
by density
gradient isolation and cryo-preserved in 50% RPMI, 40% FCS and 10% DMSO.
Samples were subsequently thawed and stained in phosphate buffered saline
(200mM
EDTA, 0.5% BSA) for surface markers (CD3, CD4, CD8, and CD62L). 1: Healthy
controls, n=73; 2: Untreated RRMS patients, n=12; 3: RRMS patients before
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14
Natalizumab therapy, n=30; 4: RRMS patients after long-term Natalizumab
therapy,
which is defined as a therapy of more than 18 months, n=78; 5: HIV patients
(CDC
stadium B1-C2), n=5; 6: HIV' patients (CDC stadium C3), n=9. White circles:
RRMS
patients under long-term Natalizumab therapy before onset of PML; Black
circles: HIV'
patients after onset of PML. The percentage of CD62L positive cells of CD3
'CD4' T
cells or CD3-tD8-' T cells is shown. An isotype control was used to define a
threshold
between CD62L positive and negative cells.
[062] Figure 4 shows the immune cell composition in peripheral blood (dots)
and CSF (triangles) of patients under Natalizumab therapy (n=18; treatment >18
months). Given are percentages of monocytes, CD4 ' and CD8' T cells and B
cells (of
total leukocytes).
[063] Figure 5 shows the in vitro migration of isolated PBMC over primary
human microvascular endothelial cells (HBMEC). Given are the absolute values
of
migrated T cells per pl of sample represented by individual dots of healthy
donors (open
circles, n=10), untreated MS patients (black, n=16) or Natalizumab patients
(grey, n=29).
Migration has been assessed after 6h.
[064] Figure 6 shows the in vitro migration of isolated PBMC over primary
human choroid plexus-derived epithelial cells (HCPEpiC). Given are the
absolute values
of migrated T cells per pl of sample represented by individual dots of healthy
donors
(open circles, n=6), untreated MS patients (black, n=6) or Natalizumab
patients (grey,
n=15). Migration has been assessed after 6h.
[065] Figure 7 shows the percentages of LFA-1 expressing T cells before
Natalizumab treatment (month 0) and in the time course of therapy (months 1,
3, 6, 12,
15-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55; n=39 patients). Black
symbols
indicate the mean calculated from patients at given time points, standard
error of the
mean are given. The white and grey circles represent two patients who later
developed
PML.
[066] Figure 8 shows the percentages of CD62L expressing T cells before
Natalizumab treatment (month 0) and in the time course of therapy (months 1,
3, 6, 12,
15-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55; n=39 patients). Black
symbols
indicate the mean calculated from patients at given time points, standard
error of the
mean are given. The white and grey circles represent two patients who later
developed
PML.
[067] Figure 9 shows the migration of CD3' T cells (in percent, related to
untreated MS patients set to 100%) before Natalizumab treatment (month 0) and
in the
time course of therapy (months 1, 3, 6, 12, 15-20, 21-25, 26-30, 31-35, 36-40,
41-45, 46-
50, 51-55; n=50 patients). Black symbols indicate the mean calculated from
patients at
given time points, standard error means are given. The white and grey circles
represent
two patients who later developed PML.
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[068] Figure 10 shows relative quantification of CD1 1 a as compared to hS18
on thawed PBMC from MS patients before (month 0) and in the time course of
therapy
(months 1, 3, 6, 12, 15-20, 21-25, 26-30, 31-40, 41-50; n=27 patients) as
assessed by
real-time PCR. Lower delta CT values indicate a higher expression of the
target.
[069] Figure 11 shows relative quantification of Runx-3 as compared to hS18
on thawed PBMC from MS patients before (month 0) and in the time course of
therapy
(months 1, 3, 6, 12, 15-20, 21-25, 26-30, 31-40, 41-50; n=28 patients) as
assessed by
real-time PCR. Lower delta CT values indicate a higher expression of the
target.
[070] Figure 12 shows relative quantification of CD62L as compared to hS18
on thawed PBMC from MS patients before (month 0) and in the time course of
therapy
(months 1, 3, 6, 12, or more; n=28 patients) as assessed by real-time PCR.
Lower delta
CT values indicate a higher expression of the target.
[071] Figure 13 depicts dot plots of samples of the six MS Natalizumab pre-
PML patients and one exemplary MS patient before the start of Natalizumab
therapy.
The numbering ofpre-PML patients is in line with the numbering used in Fig.
15. PBMC
were first gated on "live lymphocytes", then CD3 ' (T cells), then CD4 ' and
finally
plotted on CD62L vs. CD45RA to illustrate the loss of CD62L (especially
striking on the
CD45RA (naive) CD4 ' T cells).
[072] Figure 14 lists the monoclonal antibodies used for flow cytometry.
[073] Figure 15 lists all patients included in this study. Given are
cohort/patient, number of patients, year of birth, sex, first manifestation of
MS, EDSS,
pre-treatments, JCV antibody seropositivity, cycles of Natalizumab, %CD62L of
CD4 ' T
cells (mean, standard deviation, and 10-90 percentile) of the following
cohorts: Healthy
controls, MS (naïve), MS (baseline treatments), MS (Natalizumab), MS
(Natalizumab)
pre-PML, MS (Natalizumab) acute-PML, MS (Natalizumab) post-PML, other
monoclonal antibody-associated acute-PML and HIV-associated acute-PML,
corresponding to the groups in Fig. lA and Fig. 1B.
DETAILED DESCRIPTION OF THE INVENTION
[074] It must be noted that, as used herein, the singular forms "a", "an", and
"the" include plural references unless the context clearly indicates
otherwise. Thus, for
example, reference to "an antibody" includes one or more of such different
antibodies
and reference to "the method" includes reference to equivalent steps and
methods known
to those of ordinary skill in the art that could be modified or substituted
for the methods
described herein.
[075] The present invention provides methods of determining a prognosis or
the risk for PML occurrence. Using a method according to the invention a
subject can be
identified as being at a higher risk of developing PML when compared to
otherwise
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apparently similar subjects, e.g. subjects of comparable health/disease state
or risk factor
exposure. In some embodiments a method according to the invention can thus be
taken to
define a method of assessing the risk level of a subject with regard to PML
occurrence.
The invention also allows stratifying patients for risk of PML.
[076] The present invention is based on the surprising finding that CD62L
levels on T cells can be used as a biomarker for the risk evaluation of
occurrence of PML
in a subject. In part the present invention is also based on the finding that
the binding of
VLA-4 influences the expression of the cell surface molecules CD62L and LFA-1
and
basic immune cell functions such as migratory capacity. Without being bound by
any
particular theory, the present inventors have discovered that some cell
surface molecules,
including CD62L and LFA-1, are differentially expressed on T cells in subjects
who/that
develop or have developed PML. In addition, the present inventors have found
that
CD62L is already differently expressed on T cells in subjects who/that are
about to
develop PML. Use of such molecules as biomarkers, in conjunction with further
biomarkers or tests, provides an indication as to which subjects are more
likely to suffer
from PML. The biomarkers provided in the present invention can assist
physicians in
determining an appropriate therapeutic regimen.
[077] Accordingly, the biomarkers provided in the present invention can be
advantageously used to diagnose the immune competence of a subject, such as a
subject
who/ that is receiving or expected to receive long-term VLA-4 blocking agent
treatment,
a subject who/that is HIV positive as well as to diagnose the risk of the
subject to suffer
from PML.
[078] PML is a formerly rare, but severe, subacute, rapidly progressive
demyelinating disease of the brain, which was first characterized in 1958. PML
has today
reached epidemic proportions, mostly due to the fact that HIV/AIDS has
resulted in a
remarkable increase in the frequency of PML. In some locales, HIV infection
has been
found to account for more than 90% of the predisposing disorders associated
with PML.
PML is caused by lytic infection of oligodendrocytes by the John Cunningham
virus
(JCV), a double-stranded, not enveloped human polyomavirus. JCV infects
children, and
seropositivity in adults is reported to be between 50% and 60%, with higher
prevalence
in men than in women (Soelberg Sorensen, P., et al., Multiple Sclerosis
Journal (2012)
18, 2, 143-152). The nonenveloped JCV virion is taken up into cells via
clathrin
dependent receptor-mediated endocytosis. The supposedly transmittable form of
JCV has
commonly been referred to as the JCV archetype, as it has been assumed that
all other
genotypes originate from it. These assumptions, are, however, so far not
supported by
sound evidence, i.e. it is not established whether the transmittable form of
JCV is indeed
the archetypal form of the virus. It is further not known whether JCV
superinfections can
occur after initial childhood infection (White, M.K., & Khalili, K., J.
Infect. Disease
[2011] 203, 5, 578-586). PML is thought to be caused by reactivation of JCV,
which can
stay latent in a variety of tissues such as the kidneys, the tonsils, B
lymphocytes and
lymphoid organs as well as the central nervous system. Fragments of JCV DNA
have
even been found in oligodendrocytes and astrocytes in non-PML brain. The
archetypal
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form of JCV seems to be exclusively found in the kidneys of non-PML
individuals.
Pathological JCV PML-type variants, which always have, relative to the JCV
archetype,
an altered regulatory region, form in the host via an unknown mechanism.
Compared to
the JCV archetype, pathological JCV PML-type variants have also been found to
contain
in > 80% of cases an amino acid substitution in the major capsid protein, VP1,
typically
in the outer loops. Further, deletions, duplications, and point mutations in
the noncoding
regulatory region and/or the coding region, have been reported.
[079] JCV causes lytic infection and death of myelin producing
oligodendrocytes in the white matter. It also infects astrocytes in a non-
productive
fashion; an abortive infection can lead to multinucleated giant astrocytes.
PML typically
results in focal neurologic deficits such as aphasia, hemiparesis and cortical
blindness. It
is currently diagnosed by analysing cerebrospinal fluid or a brain biopsy
specimen for the
presence of JCV DNA.
[080] In the context of natalizumab treatment known risk factors for
development of PML include the duration of natalizumab exposure, prior
immunosuppressive therapy and the presence of anti-JCV antibodies (Soelberg
Sorensen
et al., 2012, supra). The elevated risks associated with prior use of
immunosuppressants,
the duration of natalizumab exposure and presence of anti-JCV antibodies
appear to be
independent of each other. The overall incidence of PML is reported to be
about two in
1000 natalizumab-treated patients (ibid.).
[081] One method of the invention is a method of diagnosing or aiding in the
diagnosis of the risk of development of a condition associated with JCV in a
subject.
JCV associated conditions and symptoms of PML generally include defects of
motor
and/or cognitive performance. Symptoms/conditions that may occur are for
instance
weakness, hemiparesis, hemiplegia, i.e. partial paralysis, ataxia, altered
mental status,
visual field disturbances including loss of vision, impaired speech including
aphasia,
cognitive deterioration, as well as the so called Alien hand syndrome.
[082] A related method of the invention is a method of diagnosing or aiding in
the diagnosis of the risk of occurrence of PML in a subject. This method of
assessing the
risk of occurrence of PML may also be taken as a method of diagnosing the
susceptibility
of the subject to PML. In this regard the term "susceptibility" as used herein
refers to the
degree of risk of a subject to an indicated condition, which may be a
pathological
condition, such as a disease or disorder. The term "susceptibility to PML"
refers to the
likelihood that PML will occur in a subject. It is understood that a
respective
diagnosis/assessment involves a valuation which may subsequently turn out to
be less
than 100 % precise for a given individual. Such assessment is in some
embodiments to
be taken as an indication of the balance of probabilities rather than as a
solid predication.
[083] A respective method according to the present invention involves analysis
of a sample from the subject in vitro. Typically the sample is, essentially
consists of, or
includes body fluid from the subject. The term "essentially consists of' is
understood to
allow the presence of additional components in a sample or a composition that
do not
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affect the properties of the sample or a composition. As an illustrative
example, a
pharmaceutical composition may include excipients if it essentially consists
of an active
ingredient.
[084] The sample may in some embodiments be one of a whole blood sample, a
blood cell sample, a lymph sample and a sample of cerebrospinal fluid. In some
embodiments the method may include providing a sample from the subject. The
sample
may have been taken at any desired point in time before carrying out the
method of the
invention. Generally time interval between taking the sample and carrying out
the
method of the invention is selected to allow analysis of viable cells. It is
within the
skilled artisan's experience to determine a respective time interval during
which T cells
in a sample can be expected to remain viable. As a general orientation, the
inventors have
found that in the form of EDTA blood, i.e. after adding a final amount of
about 1 - 2
mg/ml EDTA (typically potassium EDTA), cells remain viable and suitable for
carrying
out a method according to the invention during a time interval of up to 48
hours during
which the sample is kept in fluid form at room temperature, i.e. about 18 C.
Cells may
for instance be kept at a temperature in the range from about 2 C to about 37
C, such as
from about 4 C to about 37 C or below. In some embodiments the sample is
kept at
about 32 C or below. In some embodiments the sample is kept at a temperature
of about
25 C or below. As an illustrative example, a whole blood sample may be kept
at about
25 C or below. As a further example a cerebrospinal fluid sample may be kept
at about
25 C or below. As yet a further example a lymph sample may be kept at about
25 C or
below. In some embodiments the sample is kept at a temperature of about 22 C
or
below, such as about 18 C or below. In some embodiments the sample is kept at
about
15 C or below, such as below 10 C. In some embodiments the sample is kept at
about 4
C or at about 8 C. As an illustrative example, a whole blood sample may be
kept at
about 8 C or below. As a further example a cerebrospinal fluid sample may be
kept at
about 8 C or below.
[085] As used herein, the term "viable" refers to a cell that maintains
homeostasis by the use of one or more energy consuming mechanisms. Thus a
"viable"
cell for example includes a cell in which productive oxidative metabolism
occurs to
produce the necessary energy; a cell in which only glycolysis is used to
produce energy,
as well as a cell which maintains cellular integrity, such as the ability to
exclude, or
actively remove, certain molecules from the interior of the cell, by energy
consuming
mechanisms. In some embodiments, a viable cell is capable of undergoing
mitosis, cell
growth, differentiation, and/or proliferation. The expression "viable cell"
can be taken to
be synonymous with a "living cell", which includes a cell that is quiescent
(and thus not
going through the cell cycle), but nonetheless alive because energy production
and
consumption occurs in such a cell to maintain homeostasis.
[086] In some embodiments the sample has been taken on the same or on the
previous day, such as about 48 hours, about 42 hours, about 36 hours, about 30
hours,
about 28 hours, about 24 hours, about 18 hours, about 15 hours, about 12
hours, about 10
hours, about 8 hours, about 6 hours or less before the method of the invention
is being
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carried out. In some embodiments the sample has been taken within a period of
up to
about 48 hours, i.e. 0 to about 48 hours, to about 42 hours, to about 36
hours, to about 30
hours, to about 28 hours, to about 24 hours, to about 18 hours, to about 15
hours or 0 to
about 12 hours before the method of the invention is being carried out. The
subject, also
addressed as a patient or an individual herein, from which/whom the sample has
been
obtained is an animal, generally a mammal. The sample may be obtained or
derived from
any animal, including mammalian species such as a rabbit, a mouse, a rat, a
Guinea pig, a
hamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey, an
ape or a human.
[087] In some embodiments the sample from the individual is a frozen sample.
Generally the sample is frozen within the above detailed time intervals, e.g.
0 to about 48
or 0 to about 42 hours, and/or at the above exemplified time points, such as
about 48
hours, about 36 hours or less, after the sample has been obtained from the
individual. A
frozen sample may be formed by freezing an obtained sample after adding a
cryoprotective agent such as DMSO, glycerol and/or hydroxyethyl starch. In
some
embodiments, for instance where the sample is a blood cell sample, serum may
in
addition be added before freezing. As an illustrative example DMSO may be used
in a
final concentration in the range from about 2% to about 10 %, such as about
2%, about
4%, about 5% or about 10% DMSO. Typically the sample is then frozen at a
controlled
rate to a temperature less than -50 C, whereafter the sample may for instance
be stored,
including long-term storage, at a temperature below -130 C such as -160 C,
e.g. in liquid
nitrogen for extended periods of time.
[088] In some embodiments of a method according to the invention a sample as
provided from the individual is depleted of erythrocytes, in some embodiments
at least
essentially cleared of erythrocytes, if required. Depletion or removal of
erythrocytes may
for example be required in case the sample is a whole blood sample or a blood
cell
sample. Lysis of erythrocytes may be carried out osmotically or chemically.
Osmotic
lysis is suitable in the contest of the present invention since erythrocytes
lyse at an
osmolarity at which leukocytes remain intact. In the art typically a solution
of
ammonium chloride is used for osmotic lysis, which may further include
potassium
bicarbonate and/or EDTA. A commercially available reagent may be used, such as
the
FCM Lysing solution by Santa Cruz (order no sc-3621), Erythrolyse Red Blood
Cell
Lysing Buffer by AbD Serotec or RBC Lysis Solution by 5 PRIME. Chemical lysis
of
erythrocytes may for example be achieved using an organic solvent such as
diethylether
or chloroform, and/or a surfactant, a copper containing solution or via adding
one of
certain bacterial or animal toxins. After lysis of erythrocytes the remaining
blood cells
may be collected, for example by means of centrifugation.
[089] In a method according to the invention, the level of T cells in the
sample
that have the protein L-selectin and optionally LFA-1 on their surface is
detected. T cells
are known to the skilled artisan as lymphocytes, i.e. nucleated blood cells
that are also
called white blood cells. T cells mature in the thymus and can be
distinguished from
other lymphocytes in that they have the T cell receptor on their cell surface.
The main
known role of the T cell is recognition of antigens bound to major
histocompatibility
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complex (MHC) molecules. The T cell receptor (TCR) is a heterodimer, which
consists
of a 34 kD a-chain, linked by a disulphide bond to a 34 kD 13-chain in about
95 % of T
cells. Both chains span the plasma membrane and have accordingly an
extracellular
portion, each of which includes a variable region, termed Va and V13,
respectively.
About 5 % of T cells have a T cell receptor that consists of a y- and a 6-
chain instead of
an a- and a 13-chain, which likewise have extracellular variable regions. T
cell receptors
can, like immunoglobulins, recognize a very large number of different
epitopes.
[090] It is to be understood that "level", "value" and "amount" are used
interchangeably in the application to refer to a measurement that is made
using any
analytical method for detecting the biomarker in a sample and that indicates
the presence,
absence, absolute amount or concentration, relative amount or concentration,
titer, a
level, an expression level, a ratio of measured levels, or the like, of, for,
or corresponding
to the biomarker in the sample. The exact nature of the "value" or "level"
depends on the
specific design and components of the particular analytical method employed to
detect
the biomarker.
[091] An "epitope" is antigenic and thus an epitope may also be taken to
define
an "antigenic structure" or "antigenic determinant". Thus, a binding domain of
an
immunoglobulin or of a proteinaceous binding molecule with immunoglobulin-like
functions is an "antigen-interaction-site". The term "antigen-interaction-
site" defines, in
accordance with the present invention, a motif of a polypeptide, which is able
to
specifically interact with a specific antigen or a specific group of antigens,
e.g. L-selectin
and optionally LFA-1 in different species. This binding/interaction is also
understood to
define a "specific recognition".
[092] The term "epitope" also refers to a site on an antigen such as CD3, CD4
or CD8, with which an immunoglobulin or a proteinaceous binding molecule with
immunoglobulin-like functions forms a complex. In some embodiments, an epitope
is a
site on a molecule against which an immunoglobulin or a proteinaceous binding
molecule with immunoglobulin-like functions will be produced and/or to which
an
antibody will bind. For example, an epitope can be recognized by an
immunoglobulin or
a proteinaceous binding molecule with immunoglobulin-like functions. The
epitope may
be a "linear epitope", which is an epitope where an amino acid primary
sequence
contains the epitope recognized. A linear epitope typically includes at least
3, and more
usually, at least 5 amino acids in a unique sequence. A linear epitope may for
example
include about 8 to about 10 amino acids in a unique sequence. The epitope may
also be a
"conformational epitope", which in contrast to a linear epitope, is an epitope
where the
primary sequence of the amino acids that includes the epitope is not the sole
defining
component of the epitope recognized (e.g., an epitope wherein the primary
sequence of
amino acids is not necessarily recognized by the antibody defining the
epitope).
Typically a conformational epitope includes a larger number of amino acids
than a linear
epitope. With regard to recognition of conformational epitopes, an
immunoglobulin or a
proteinaceous binding molecule with immunoglobulin-like functions recognizes a
3-
dimensional structure of the antigen, such as a peptide or protein or a
fragment thereof.
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As an illustrative example, when a protein molecule folds to form a three
dimensional
structure, certain amino acids and/or all or portions of the polypeptide
backbone forming
the conformational epitope become juxtaposed, allowing an antibody to
recognize the
epitope. Methods of determining conformation of epitopes include, but are not
limited to,
x-ray crystallography, 2-dimensional nuclear magnetic resonance spectroscopy,
site-
directed spin labelling and electron paramagnetic resonance spectroscopy.
[093] In some embodiments the presence of the T cell receptor on the surface
of a cell may be used to identify the cell as a T cell. As the T cell receptor
has variable
regions it may, nevertheless, be advantageous to use another cell surface
protein to
identify a T cell. An example of suitable protein in this regard is a T cell
co-receptor.
Two illustrative examples of a co-receptor of the T cell receptor are the
protein complex
CD3 (Cluster of Differentiation 3) and the protein CD247. CD3 has four chains,
which
are in mammals one D3y chain, one CD3 6 chain, and two CD38 chains. These
chains
associate with a molecule known as the T-cell receptor and at least one T-cell
surface
glycoprotein CD3 zeta chain also known as T-cell receptor T3 zeta chain or
CD247
(Cluster of Differentiation 247). CD247 may be present on the cell surface as
either a c2
complex or a c/ri complex. The complex of TCR, CD247 and CD3 can generate an
activation signal in T lymphocytes. The TCR, c-chain(s), and CD3 molecule
together
define the TCR complex. In practicing a method according to the invention
identifying
the presence of CD3 on a particular cell or plurality of cells is often a
convenient way of
identifying T cells. Therefore the terms "CD3 ' T cell" and "T cell" are used
interchangeable herein to address a T cell and to distinguish a T cell from
other cell
types.
[094] A further example of a co-receptor of the T cell receptor is the
transmembrane protein CD8 (Cluster of Differentiation 8). Most T cells that
have CD8
on their surface are cytotoxic T cells. CD8 plays an important role in binding
to the class
I major histocompatibility complex. Two isoforms of the protein, namely CD8-
alpha and
-beta, are known. Each such chain contains a domain that resembles an
immunoglobulin
variable domain. CD8 is a dimer of two of these chains, either a homo- or a
heterodimer.
[095] CD4 ' T cells in addition to CD3 have the CD4 (Cluster of
Differentiation
4) protein on their surface, a glycoprotein consisting of four extracellular
immunoglobulin domains, termed D1 to D4, and a small cytoplasmic region. The
CD4
protein is known to be used by HIV-1 to gain entry into T cells of a host. CD4
' T cells
can be classified into a variety of cell populations with different functions
and should
thus not be taken to define a unitary set of cells. Typical examples of a CD4
' T cell are a
T helper cell, a regulatory T cell and a memory T cell.
[096] In some embodiments a method according to the invention includes
identifying CD3 ' T cells in the sample, for example by employing an
immunoglobulin,
an immunoglobulin fragment or a proteinaceous binding molecule with antibody-
like
functions as further explained below. By "fragment" in reference to a
polypeptide such
as an immunoglobulin or a proteinaceous binding molecule is meant any amino
acid
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sequence present in a corresponding polypeptide, as long as it is shorter than
the full
length sequence and as long as it is capable of performing the function of
interest of the
protein ¨ in the case of an immunoglobulin specifically binding to the desired
target, e.g.
antigen (CD62L and optionally LFA-1 , for example). The term "immunoglobulin
fragment" refers to a portion of an immunoglobulin, often the hypervariable
region and
portions of the surrounding heavy and light chains that displays specific
binding affinity
for a particular molecule. A hypervariable region is a portion of an
immunoglobulin that
physically binds to the polypeptide target.
[097] In some of these embodiments identifying CD3 ' T cells in the sample
serves in distinguishing CD3 ' T cells from other cells such as CD3- cells or
non-T cells.
In some embodiments CD4 T cells are identified in the sample. Identifying CD4
' T cells
typically serves in distinguishing CD4' T cells from other cells such as CD4-
T cells or
non-T cells. In some embodiments CD8' T cells are identified in the sample.
Identifying
CD8' T cells typically serves in distinguishing CD8' T cells from other cells
such as
CD8- T cells or non-T cells. It is understood that CD4' T cells and CD8' T
cells are
typically also CD3 ' T cells so that a CD3 ' T cell identified may also for
instance be a
CD4' T cell rather than be distinguished from a CD4' T cell. Accordingly, in
some
embodiments in a first step a T cell may be identified as a CD3 ' T cell. In a
second step
it may be determined whether the CD3 ' T cell is a CD4' T cell. It may also be
determined whether the CD3 ' T cell is a CD8' T cell. In some embodiments T
cells are
identified by the presence of CD3. Of the thus identified T cells CD4-' T
cells are
distinguished from CD8-' T cells.
[098] In some embodiments a method according to the invention includes
enriching and/or isolating CD3 ' T cells from the sample. In some embodiments
a method
according to the invention includes enriching and/or isolating CD4' T cells
and/or CD8'
T cells from the sample. In some embodiments T cells are enriched, including
sorted,
based on the presence of CD3 on the cell surface. Of the thus enriched T
cells, those T
cells that have CD4 on their surface, i.e. CD4' T cells, may be further
enriched. In some
embodiments, of the T cells that have been enriched based on the presence of
CD3, those
T cells that have CD8 on their surface, i.e. CD8' T cells, may be further
enriched. As an
illustrative example, the sample may be from an individual undergoing
treatment with a
VLA-4 blocking agent. CD3 ' T cells may be enriched in a first step, of which
CD4' T
cells may be enriched in a second step, thereby obtaining enriched CD3 'CD4' T
cells.
The CD3 'CD4' T cells of the individual undergoing treatment with a VLA-4
blocking
agent may then be used in a method according to the invention. Furthermore,
CD8' T
cells may be enriched in a second step, thereby obtaining enriched CD3-tD8-' T
cells
from the individual undergoing treatment with a VLA-4 blocking agent. CD8
positive T
cells or CD4 positive T cells of the individual may for instance be used to
analyse the
expression of CD62L thereon.
[099] In some embodiments enriching and/or isolating CD3 ' T cells, CD4' T
cells and/or CD8' T cells from the sample includes cell sorting and/or
selection, for
instance via negative magnetic immunoadherence or flow cytometry. In some
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23
embodiments enriching and/or isolating such cells consist of cell sorting or
selection.
Such a technique may be based on contacting the cells with a plurality of
antibodies
directed to cell surface markers present on the cells negatively selected. As
an illustrative
example, to enrich for CD4 ' cells by negative selection, a plurality of
antibodies may
include antibodies directed to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8,
while to
enrich for CD8 cells by negative selection, a plurality of antibodies may
include
antibodies directed to CD14, CD20, CD1 lb, CD16, and HLA-DR.
[0100] In some embodiments it may be desired to enrich for or positively
select
for T cells that express CD3'. In some embodiments undesired cells are
depleted by
contacting them with particles/beads on which antibodies are immobilized that
bind to
proteins found on undesired cells, but not on desired cells. In some
embodiments desired
cells are collected from the sample by contacting them with beads on which
antibodies
are immobilized that bind to proteins found on the desired cells, but not on
undesired
cells.
[0101] For isolation of a desired population of cells by positive or negative
selection, the amount and concentration of cells and particle/bead surface can
be varied.
In certain embodiments it may be desired to reduce the volume in which beads
and cells
are contacted, for instance to ensure maximum contact of cells and beads. For
example,
in one embodiment, a concentration of about 2 billion cells/ml is used. In one
embodiment, a concentration of about 1 billion cells/ml is used. In a further
embodiment,
a concentration of more than about 100 million cells/ml is used. In some
embodiments a
concentration of cells of about 10 million cells/ml or more is used. In some
embodiments
cells are at a concentration of about 15, including about 20, about 25 or
about 30 million
cells/ml. In some embodiments a concentration of cells of about 35, about 40,
about 45,
about 50 million cells/ml or more is used. In some embodiments a concentration
of cells
of about 75 million cells/ml is used. In some embodiments cells are at a
concentration of
about 80 million cells/ml. In some embodiments cells are at a concentration of
about 85
million cells/ml. The concentration of cells may for example be about 90,
including
about 95, about 100, or about 125 million cells/ml or more. In some
embodiments a
concentration of cells of about 150 million cells/ml or more is used. The use
of high cell
concentrations may in some embodiments result in increased cell yield, cell
activation,
and cell expansion. In some embodiments the use of high cell concentrations
may allow
more efficient capture of cells that may express e.g. CD62L in low number.
[0102] The word "about" as used herein refers to a value being within an
acceptable error range for the particular value as determined by one of
ordinary skill in
the art, which will depend in part on how the value is measured or determined,
i.e., the
limitations of the measurement system. For example, "about" can mean within 1
or more
than 1 standard deviation, per the practice in the art. The term "about" is
also used to
indicate that the amount or value in question may be the value designated or
some other
value that is approximately the same. The phrase is intended to convey that
similar
values promote equivalent results or effects according to the invention. In
this context
"about" may refer to a range above and/or below of up to 10%. The word "about"
refers
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in some embodiments to a range above and below a certain value that is up to
5%, such
as up to up to 2%, up to 1%, or up to 0.5 % above or below that value. In one
embodiment "about" refers to a range up to 0.1 % above and below a given
value.
[0103] Those skilled in the art will recognize or be able to ascertain, using
no
more than routine experimentation, many equivalents to the specific
embodiments of the
invention described herein. Such equivalents are intended to be encompassed by
the
present invention. Throughout this specification and the claims which follow,
unless the
context requires otherwise, the word "comprise", and variations such as
"comprises" and
"comprising", will be understood to imply the inclusion of a stated integer or
step or
group of integers or steps, but not the exclusion of any other integer or step
or group of
integer or step. Several documents are cited throughout the text of this
specification.
Each of the documents cited herein (including all patents, patent
applications, scientific
publications, manufacturer's specifications, instructions, etc.) is
incorporated by
reference in their entirety. Nothing herein is to be construed as an admission
that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
[0104] Progressive multifocal encephalopathy is a fatal demyelinating disease
of
the central nervous system (CNS), caused by the lytic infection of
oligodendrocytes by a
human polyomavirus, JC virus (JCV). PML is rare disease which mostly develops
in
patients with underlying immunosuppressive conditions, including Hodgkin's
lymphoma, lymphoproliferative diseases and in those undergoing antineoplastic
therapy
and AIDS. As discussed earlier, PML is also associated with demyelinating
disease or
autoimmune disease patients including multiple sclerosis and Crohn's disease
who are
treated with antibody-based regimens (natalizumab, efalizumab and rituximab).
This
indicates the existence of a strong link between the underlying
immunosuppressive
conditions and development of PML. MRIs of the brain can detect active disease
and, in
some cases, in the preclinical state. However, given the rapid progression of
PML, scans
would have to be taken frequently to provide hope of earlier detection. Since
PML is a
potentially fatal disease with no specific therapy available, there is a
pressing need for a
method screening for patients at increased risk for PML.
[0105] To address this need, the present invention provides a method of
assessing an individual's immune competence and susceptibility to PML. This is
also
described herein as assessing the "risk of occurrence" of PML for an
individual. As used
herein, the term "susceptibility" or "risk of occurrence" refers to the degree
of risk of a
subject to a given disease or pathological condition. The term susceptibility
or risk of
occurrence to PML refers to the likelihood of a subject to develop or suffer
from PML,
particularly if the patient continues with the treatment. As will be
understood by those
skilled in the art, such determination is usually not intended to be correct
for 100% of the
subjects to be analyzed.
[0106] According to the present invention, the term "patient" or "subject"
refers
to animals, preferably mammals, and more preferably, humans. For the purpose
of the
present application, a "patient undergoing VLA4 blocking agent treatment" is
defined as
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a patient who is expected to receive, receiving or having received VLA-4
blocking
agent. VLA-4 (Very Late Antigen-4, also termed Integrin a4131) is expressed on
the
surface of all leukocytes except neutrophils. It recognizes the vascular cell
adhesion
molecule-1(VCAM-1), an inducible cell surface molecule which mediates the
adhesion
of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium.
VCAM-
1 binds to the VLA-4 when the leukocytes are activated by chemotactic agents
or other
stimuli, and mediates leukocyte extravasion to sites of tissue inflammation.
Structurally,
VLA-4 is a heterodimer composed of CD49d (a4) and CD29 (131) (Hemler et al,
"Structure of the Integrin VLA-4 and Its Cell-Cell and Cell Matrix Adhesion
Functions,"
1990, Immunological Reviews, 114:45-65.
[0107] A VLA-4 blocking agent is defined as a molecule which binds to VLA-4
antigen on the surface of a leukocyte with sufficient specificity to inhibit
the VLA-
4NCAM-1 interaction. In a preferred embodiment, the blocking agent binds to
VLA-4
integrin with a Kd of less than 10-6 M. VLA-4 blocking agents may be a VLA-4
binding
antibody (a full length VLA-4 binding antibody) or VLA-4 binding antibody
fragment,
such as a Fab fragment, a F(ab')2 fragment, a bivalent fragment including two
Fab
fragments linked by a disulfide bridge at the hinge region, a Fd fragment
consisting of
the VH and CHI domains, a Fv fragment consisting of the VL and VH domains of a
single arm of an antibody, a dAb fragment which consists of a VH domain), or
an
isolated complementarity determining region (CDR) that retains functionality.
[0108] VLA-4 blocking agents inhibit the migration of leukocytes from the
blood to the central nervous system by disrupting adhesion between the T-cell
and
endothelial cells. This is believed to result in the reduction of
proinflammatory cytokines,
and thus the reduction of the occurrence of pathologic inflammatory disease
within the
CNS.
[0109] VLA-4 blocking agents include, besides Natalizumab (Biogen U.S. Pat.
No. 5,840,299), MAbs HP2/1, HP1/3 (Elices et al, 1990, "VCAM-1 on Activated
Endothelium Interacts with the Leukocyte Integrin VLA-4 at a Site Distinct
from the
VLA-4/Fibronectin Binding Site", Cell 60:577-584), HP1/2 (Sanchez-Madrid et
al, 1986,
"VLA-3: A novel polypeptide association within the VLA molecular complex: cell
distribution and biochemical characterization," Eur. J. Immunol, 16:1343-
1349),
humanized HP1/2 (U.S. Pat. No. 6,602,503), HP1/7, HP2/4, B-5G10, T52/16
(Pulido et
al, 1991, "Functional Evidence for Three Distinct and Independently
Inhibitable
Adhesion Activities Mediated by the Human Integrin VLA-4," J Biol. Chem.
266(16):10241-5), mAB L25 (Becton Dickinson GmBH, Germany), P4C2 (Abeam,
Cambridge, UK), and AJM300 (Ajinomoto, Japan), and recombinant anti-VLA4
antibodies as described in U.S. Pat. No. 6,602,503 and U.S. Pat. No.
7,829,092. one
embodiment, the VLA-blocking agent is CD49d(a4) specific.
[0110] Furthermore, the VLA-4 blocking agents may also be VLA-4 antagonists
that are not monoclonal antibodies, such as SB-683699 (GlaxoSmithKline,
Middlesex,
UK),a small molecule dual a4 antagonist and small molecules CS-1
peptidomimetics
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26
(U.S. Pat. Nos. 5,821,231, 5,869,448, 5,869,448; 5,936,065; 6,265,572;
6,288,267;
6,365,619; 6,423,728; 6,426,348; 6,458,844; 6,479,666; 6,482,849; 6,596,752;
6,667,331; 6,668,527; 6,685,617; 6,903,128; and 7,015,216), phenylalanine
derivatives
(U.S. Pat. Nos. 6,197,794; 6,229,011; 6,329,372; 6,388,084; 6,348,463;
6,362,204;
6,380,387; 6,445,550; 6,806,365; 6,835,738; 6,855,706; 6,872,719; 6,878,718;
6,911,451; 6,916,933; 7,105,520; 7,153,963; 7,160,874; 7,193,108; 7,250,516;
and
7,291,645) alphafeto protein (U.S. Pat. Pub. No. 2010/0150915), Beta-amino
acid
compounds (U.S. Pat. Pub. Nos. 2004/0229859 and 2006/0211630), semi-peptide
compounds (U.S: Pat. No. 6,376,538), Leu-Asp-Val tripeptide (U.S. Pat. No.
6,552,216),
pegylated molecules as described in (U.S. Pat. Pub. No. 2007/066533, U.S. Pat.
No.
6,235,711).
[0111] In some embodiments, the patient is diagnosed with multiple sclerosis,
including relapsing-remitting multiple sclerosis (RRMS) and secondary
progressive
multiple sclerosis (SPMS), as well as Crohn's disease and other autoimmune
diseases.
The term "treatment" as used herein, means to reduce, stabilize, or inhibit
progression of
a symptom which are associated with the presence and/or progression of a
disease or
pathological condition
[0112] Isolation of a desired population of cells may in some embodiments
include general cell enrichment techniques such as centrifugation, filtration
or cell
chromatography. Generally, isolating or enriching a desired population of
cells may be
carried out according to any desired technique known in the art. In some
embodiments
isolation of a desired population of cells may include the use of a
commercially available
cell isolation kit. T cells may for instance be obtained from peripheral
blood, from blood,
cerebrospinal fluid, or enriched fractions thereof. T cells may for instance
be obtained
from peripheral blood mononuclear cells (PBMC) such as human PBMCs. In some
embodiments PBMC may for instance be enriched using a standard technique based
on
cell density and/or cell size. As an illustrative example, PBMC may be
enriched or
isolated via density gradient centrifugation, for example using sucrose,
dextran, Fico11
or Perco11 . T cells may then be enriched or purified from the obtained PBMCs,
for
example using a commercially available T cell isolation kit such as the
Dynabeads0
UntouchedTM Human CD4 T Cells kit available from Invitrogen or the StemSep
Human CD4 T Cell Enrichment Kit from STEMCELL Technologies Inc..
[0113] The term "purified" is understood to be a relative indication in
comparison to the original environment of the cell, thereby representing an
indication
that the cell is relatively purer than in the natural environment. It
therefore includes, but
does not only, refer to an absolute value in the sense of absolute purity from
other cells
(such as a homogeneous cell population). Compared to the natural level, the
level after
purifying the cell will generally be at least 2-5 fold greater (e.g., in terms
of cells/m1).
Purification of at least one order of magnitude, such as about two or three
orders,
including for example about four or five orders of magnitude is expressly
contemplated.
It may be desired to obtain the cell at least essentially free of
contamination, in particular
free of other cells, at a functionally significant level, for example about
90%, about 95%,
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27
or 99% pure. With regard to a nucleic acid, peptide or a protein, the above
applies
mutatis mutandis. In this case purifying the nucleic acid, peptide or protein
will for
instance generally be at least 2-5 fold greater (e.g., in terms of mg/ml).
[0114] The term "isolated" indicates that the cell or cells or the peptide(s)
or
nucleic acid molecule(s) has/have been removed from its/their normal
physiological
environment, e.g. a natural source, or that a peptide or nucleic acid is
synthesized. Use of
the term "isolated" indicates that a naturally occurring sequence has been
removed from
its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be
in a cell-
free solution or placed in a different cellular environment. Thus, a cell or
cells may be
included in a different medium such as an aqueous solution than provided
originally, or
placed in a different physiological environment. By "isolated" in reference to
a
polypeptide or nucleic acid molecule is meant a polymer of amino acids (2 or
more
amino acids) or nucleotides coupled to each other, including a polypeptide or
nucleic
acid molecule that is isolated from a natural source or that is synthesized.
The term
"isolated" does not imply that the sequence is the only amino acid chain or
nucleotide
chain present, but that it is essentially free, e.g. about 90 - 95% pure or
more, of e.g. non-
amino acid material and/or non-nucleic acid material, respectively, naturally
associated
with it.
[0115] By the use of the term "enriched" in reference to a polypeptide, a
nucleic
acid or a cell is meant that the specific amino acid/nucleotide sequence or
cell, including
cell population, constitutes a significantly higher fraction (2 - 5 fold) of
the total amino
acid sequences or nucleic acid sequence present in the sample of interest than
in the
natural source from which the sample was obtained. The polypeptide, a nucleic
acid or a
cell may also constitute a significantly higher fraction than in a normal or
diseased
organism or than in normal or diseased cells or in the cells from which the
sequence was
taken. This could be caused by preferential reduction in the amount of other
amino
acid/nucleotide sequences or cells present, or by a preferential increase in
the amount of
the specific amino acid/nucleotide sequence or cell of interest, or by a
combination of the
two. However, it should be noted that enriched does not imply that there are
no other
amino acid sequences, nucleotide sequences or cells present. The term merely
defines
that the relative amount of the sequence of interest has been significantly
increased. The
term significant here is used to indicate that the level of increase is useful
to the person
achieving such an increase, and generally means an increase relative to other
amino acid
sequences of about at least 2-fold, for example at least about 5- to 10-fold
or even more.
The term is meant to cover only those situations in which man has intervened
to increase
the proportion of the desired amino acid sequence, nucleotide sequence or
cell.
[0116] Where desired, further matter may be added to the sample for analysis,
for example dissolved or suspended in the sample. It is understood that any
dilution due
to such addition of matter has to be accounted for and may need to be
considered when
calculating the level of L-selectin (CD62L) expressing T cells, including CD4
' or CD8'
T cells. As an illustrative example one or more buffer compounds may be added
to the
sample. Numerous buffer compounds are used in the art and may be used to carry
out the
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various methods described herein. Examples of buffers include, but are not
limited to,
solutions of salts of phosphate, carbonate, succinate, carbonate, citrate,
acetate, formate,
barbiturate, oxalate, lactate, phthalate, maleate, cacodylate, borate, N-(2-
acetamido)-2-
amino-ethanesulfonate (also called (ACES), N-(2-hydroxyethyl)-piperazine-N'-2-
ethanesulfonic acid (also called HEPES), 4-(2-hydroxyethyl)-1-piperazine-
propanesulfo-
nic acid (also called HEPPS), piperazine-1,4-bis(2-ethanesulfonic acid) (also
called
PIPES), (24tris(hydroxymethyl)-methylamino]-1-ethansulfonic acid (also called
TES),
2-cyclohexyl-amino-ethansulfonic acid (also called CHES) and N-(2-acetamido)-
iminodiacetate (also called ADA). Any counter ion may be used in these salts;
ammonium, sodium, and potassium may serve as illustrative examples. Further
examples
of buffers include, but are not limited to, triethanolamine, diethanolamine,
ethylamine,
triethylamine, glycine, glycylglycine, histidine,
tris(hydroxymethyl)aminomethane (also
called TRIS), bis-(2-hydroxyethyl)-imino-tris(hy-droxymethyOmethane (also
called BIS-
TRIS), and N-rfris(hydroxymethyl)-methyl]glycine (also called TRICINE), to
name a
few. A respective buffer may be an aqueous solution of such buffer compound or
a
solution in a suitable polar organic solvent. Further examples of matter that
may be
added to the sample include salts, detergents or chelating compounds. As yet a
further
illustrative example, nuclease inhibitors may need to be added in order to
maintain a
nucleic acid molecule in an intact state.
[0117] In an embodiment of a method according to the invention the level of L-
selectin (CD62L) expressing CD3 ' T cells in the sample is detected. The terms
"expression", "gene expression" or "expressing" as used herein relate to the
entirety of
regulatory pathways converting the information encoded in the nucleic acid
sequence of
a gene first into messenger RNA (mRNA) and then to a protein. Accordingly, the
expression of a gene includes its transcription into a primary hnRNA, the
processing of
this hnRNA into a mature RNA and the translation of the mRNA sequence into the
corresponding amino acid sequence of the protein. In this context, it is also
noted that the
term "gene product" refers not only to a protein, including e.g. a final
protein (including
a splice variant thereof) encoded by that gene and a respective precursor
protein where
applicable, but also to the respective mRNA, which may be regarded as the
"first gene
product" during the course of gene expression.
[0118] The protein L-selectin may be any respective variant or isoform of the
respective species, e.g. human. The protein may for example be the human
protein of the
Swissprot/Uniprot accession number P14151 (version 145 as of 22 February 2012)
or the
human protein of the Swissprot/Uniprot accession number Q9UJ43 (version 97 as
of 22
February 2012). This protein may for instance be encoded by the SELL gene of
GenBank
accession number NG 016132 (version NG 016132.1 as of 01 February 2012;
GI:270047500). The protein may for example be encoded by the mRNA of GenBank
accession number BCO20758 (version BCO20758.1 as of 04 August 2008; GI:
18088807). The protein may in some embodiments be the mouse protein of the
Swissprot/ Uniprot accession number P18337 (version 116 as of 22 February
2012). In
some embodiments the protein may be the rat protein of the Swissprot/Uniprot
accession
number P30836 (version 94 as of 22 February 2012) or the rat protein of the
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29
Swissprot/Uniprot accession number Q63762 (version 89 as of 22 February 2012).
The
protein may also be the bovine protein of the Swissprot/Uniprot accession
number
P98131 (version 82 as of 22 February 2012) or the bovine protein of the
Swissprot/Uniprot accession number F1N4U9 (version 8 as of 22 February 2012).
[0119] As discussed earlier, PML is observed in patients administered with the
VLA-4 blocking agent Natalizumab. It is still unclear how such treatment
interferes with
JC virus control. Due to the few biomarkers available to identify patients at
risk for
developing PML, and that the benefit of such treatment outweighs the risk in
most cases,
there is an urgent need for a test which preemptively stratifies patients who
are more
prone to PML. As used herein, a "biomarker" is a substance or a condition
whose
detection indicates a particular biological state, such as a weakened immune
competence.
[0120] From another perspective, the present invention provides a method of
screening patients who are more susceptible to PML. The term "screen" or
"screening" as
used herein refers to the process of determining the susceptibility,
propensity, risk, or
risk assessment of a subject for having or developing a disorder. The method
of the
present invention comprises monitoring the biomarker expressed in a sample
from a
patient. The term "monitor," as used herein, generally refers to the
overseeing,
supervision, regulation, watching, tracking or surveillance of an activity.
For example,
the term "monitoring the expression of CD62L" refers to measuring and
following the
expression of mRNA or protein of CD62L over time at predetermined time
intervals.
[0121] The present invention provides a novel biomarker for the risk
evaluation
for patients undergoing VLA-4 blocking agent. The inventors have surprisingly
discovered that the change in the level of mRNA or protein expression of the
biomarkers
presently provided can be associated with a weakened immune state and a
susceptibility
to PML. One of the novel biomarkers is CD62L protein, also known as L-selectin
and
SELL. It is a cell adhesion molecule found on leukocytes which has been
described to be
one of the key molecules involved in the homing of T cells to the secondary
lymphoid
organs. CD62L mediates lymphocyte homing to high endothelial venules of
peripheral
lymphoid tissue and leukocyte rolling on activated endothelium at inflammatory
sites.
Most peripheral blood B cells, T cells, monocytes and granulocytes express
CD62L/L-
selectin. However, some natural killer cells, spleen lymphocytes, bone marrow
lymphocytes, bone marrow myeloid cells, thymocytes, and certain hematopoietic
malignant cells also express CD62L. Its expression is commonly used to
differentiate
between central- and effector-memory T cells.
[0122] The expression of the biomarkers disclosed herein can be measured prior
to, at the same time, or at an early stage of the treatment of VLA-4 blocking
agents. To
monitor the expression of the biomarker, a sample is first isolated from the
patient. The
sample may be any bodily fluid. Preferably, peripheral blood is used so
mononuclear
blood cells can be obtained. Peripheral mononuclear blood cells can be
isolated using
any techniques known to a skilled person in the art, such as density gradient
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centrifugation using lymphocyte separation medium (PAA Laboratories, Pasching,
Austria) or Ficoll-Paque (Amerisham biosciences, Uppsala, Sweden).
[0123] It is to be understood that the term "expression" as used herein refers
to
the transcription from a gene to give an RNA, as well as the translation from
the RNA
molecule to give a protein.
[0124] The processes of isolating T cells are not limited to any particular
technique. Preferably, T cells can be isolated depending upon cell density,
affinity of
antibody against cell surface epitope, cell size, and/or degree of fluorescent
emission. For
example, T cells may be isolated by conducting a density gradient
centrifugation using
albumin, dextran, Ficoll, metrizamid, Percoll, MACS (Miltenyi Biotec,
Bergisch
Gladbach, Germany) and further identified using appropriate antibodies, such
as anti-
CD4 T cell antibody. Centrifugal elutriation, cell size, FACS using
fluorescence,
magnetic bead isolation or other methods known in the art may also be used.
[0125] Expression may be identified by any method known in the art. Needless
to say, the expression monitored can be, for example, mRNA expression or
protein
expression of the biomarkers. Any method for determining biomarker expression
may be
used to compare the expression level. Suitable methods include immunoassays,
such as
competitive and non-competitive assay systems, using techniques such as
Western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin
reactions, immunodiffusion assays, agglutination assays, complement-fixation
assays,
immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays.
The assays will develop a signal which is indicative for the presence or
absence of the
biomarkers. The signal strength can be correlated directly or indirectly (for
example,
reverse-proportional) to the amount of polypeptide present in a sample. Other
suitable
method measuring a physical or chemical property specific for the protein is
precise
molecular mass or NMR spectrum. The methods may include, biosensors, optical
devices
coupled to immunoassays, biochips, analytical devices such as mass-
spectrometers,
NMR-analyzers, or chromatography devices. Further methods include microplate
ELISA-based methods, fully automated or robotic immunoassays (such as ELECSYS
analyzers from Roche Diagnostics), CBA (an enzymatic cobalt binding assay) and
latex
agglutination assays.
[0126] Provided herein is also the use of mRNAs as biomarkers to monitor the
patient's immune competence during the course of treatment with VLA-4 blocking
agents. For example, the mRNA level of CD62L and optionally LFA-1 can be used
to
determine whether the treatment might lead to the development of PML. The
method
provided herein is useful for the initial evaluation of whether to such
treatment should be
pursued. The method is also useful for following the progress of the treatment
and
enabling a timely withdrawl, should any loss or deterioration of immune
incometence is
detected.The biomarkers of the present invention may also be used to track the
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31
effectiveness of the treatment and to gather information needed to make
adjustments in
the patient's treament by, for example, decreasing or even increasing the dose
as needed.
[0127] The determination of expression may be based on the normalized
expression level of the biomarkers. Expression levels are normalized by
correcting the
absolute expression level of a biomarker by comparing its expression to the
expression of
a gene that is not a biomarker. Alternatively, the expression level can be
provided as a
relative expression level.
[0128] In an embodiment of a method according to the invention, the level of
LFA-1 expressing T cells in the sample is detected. LFA-1 is an integrin-type
cell
adhesion molecule that is predominantly involved in leukocyte trafficking and
extravasation. LFA-1 binds to CD54, the Intercellular Adhesion Molecule 1, on
antigen-
presenting cells. LFA-1 is a heterodimer having a 13-chain, termed CD18, and
an a-chain,
termed CD1 1 a. Both the cc-chain and the 13-chain contain a von Willebrand
factor type A
domain (VWFA domain) in their N-terminal portion, also called inserted domain
(I-
domain), that plays a central role in regulating ligand binding. Also known as
CD11a/CD18, LFA-1 plays a crucial role in many cellular and immunological
processes
(migration, antigen presentation, cytotoxicity, cell proliferation and
haematopoiesis) by
displaying both signaling and adhesive properties.
[0129] The protein CD18 may be any respective variant or isoform of the
respective species, e.g. human. In some embodiments CD18 is the human protein
of the
Swissprot/Uniprot accession number Swissprot/Uniprot accession number P05107
(version 164 as of 22 February 2012) or the human protein of the
Swissprot/Uniprot
accession number B4E021 (version 24 as of 22 February 2012). The protein may
also be
the goat protein of the Swissprot/Uniprot accession number Q5VI41 (version 40
as of 25
January 2012), the porcine protein of the Swissprot/Uniprot accession number
P53714
(version 85 as of 22 February 2012) or the bovine protein of the
Swissprot/Uniprot
accession number P32592 (version 101 as of 22 February 2012). CD18 may be the
protein encoded by the ITGB2 gene, for example the mouse gene of GenBank Gene
ID
No 12575 as of 19 February 2012 or the human gene of GenBank Gene ID No 3689
as of
19 February 2012.
[0130] The protein CD45 may be any respective variant or isoform of the
respective species, e.g. human. The protein may for example be the human
protein of the
Swissprot/Uniprot accession number P20701 (version 137 as of 22 February 2012)
or the
human protein of the Swissprot/Uniprot accession number Q96HB1 (version 76 as
of 22
February 2012). The protein may also be the mouse protein of the
Swissprot/Uniprot
accession number P24063 (version 108 as of 22 February 2012) or the bovine
protein of
the Swissprot/Uniprot accession number P61625 (version 56 as of 22 February
2012).
CD45 may be the protein encoded by the ITGAL gene, such as the human gene of
GenBank Gene ID No 3683 as of 05 February 2012, the bovine gene of GenBank
Gene
ID No 281874 as of 04 February 2012 or the mouse gene of GenBank Gene ID No
16408
as of 14 February 2012.
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[0131] The word "detect" or "detecting" refers to any method that can be used
to
detect the presence of a nucleic acid (DNA and RNA) or a protein/polypeptide.
When
used herein in combination with the words "level", "amount" or "value" the
word
"detect" or "detecting" is understood to generally refer to a quantitative
rather than a
qualitative level. Accordingly, the method includes a quantification of CD62L
and
optionally LFA-1- i.e. the amount or number of CD62L expressing and optionally
LFA-1
expressing T cells, e.g. CD3 positive T cells, is determined. In this regard
the words
"value," "amount" and "level" are used interchangeably herein. The terms
"value,"
"amount" and "level" also refer to the rate of synthesis of CD62L and
optionally LFA-1
in CD3 T cells, as explained further below. The rate of synthesis of CD62L may
for
example be assessed by determining the synthesis rate of messenger RNA (mRNA)
encoded by the selectin L (SELL) gene. Synthesis of CD62L mRNA refers to any
mRNA
transcribed from a SELL gene (e.g. GenBank accession No. NG_016132, version
NG 016132.1, GI: 70047500). Currently two transcript variants of human SELL
are
known, termed variant 1 (GenBank accession No. NM 000655, version NM 000655.4,
GI:262206314) and variant 2 (GenBank accession No. NR_029467, version
NR 029467.1; GI:262205323). Synthesis of CD18 mRNA refers to any mRNA
transcribed from a ITGB2 gene. Synthesis of CD45 mRNA refers to any mRNA
transcribed from a ITGAL gene.
[0132] The rate of synthesis of LFA-1 may in some embodiments be detected by
determining the synthesis rate of mRNA encoded by the ITGAL gene and the ITGB2
gene. Synthesis of ITGAL mRNA refers to any mRNA transcribed from an ITGAL
gene.
Currently two transcript variants of the human integrin alpha L gene are
known, termed
variant 1 (GenBank accession No. NM 002209, version NM 002209.2, GI:167466214)
and variant 2 (GenBank accession No. NM 001114380, version NM 001114380.1;
GI:167466216). Human mRNA of the human ITGAL gene may also have or include the
sequence of GenBank accession No. BC008777 (version BC008777.2, GI:33870544).
Four transcript variants of the mouse ITGAL gene are known, termed variant 1
(GenBank accession No. NM 001253872, version NM 001253872.1, GI:359751454),
variant 2 (GenBank accession No. NM 008400, version NM 008400.3;
GI:359751456),
variant 3 (GenBank accession No. NM 001253873, version NM 001253873.1;
GI:359751457) and variant 4 (GenBank accession No. NM 001253874, version
NM 001253874.1; GI:359751459). Further illustrative examples of ITGAL mRNA the
synthesis rate of which may be analyzed, are dog mRNA with the sequence of
GenBank
accession No. XM 547024 (version XM 547024.2, GI:73958404), wild boar mRNA
with the sequence of GenBank accession No. EF585976 (version EF585976.1,
GI:156601155) and rat mRNA with the sequence of GenBank accession No. BC101849
(version BC101849.1, GI:74353690).
[0133] Synthesis of ITGB2 mRNA refers to any mRNA transcribed from an
ITGB2 gene. Currently two transcript variants of the human integrin beta 2
gene are
known, termed variant 1 (GenBank accession No. NM 000211, version NM 000211.3,
GI:188595673) and variant 2 (GenBank accession No. NM 001127491, version
NM 001127491.1; GI:188595676). Human mRNA of the human ITGAL gene may also
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33
have or include the sequence of GenBank accession No. S75297 (version
S75297.1;
GI:242219). Further examples of ITGB2 mRNA, the synthesis of which may be
determined, include, but are not limited to, mouse mRNA with the sequence of
GenBank
accession No. NM 008404 (version NM 008404.4, GI:145966904), rat mRNA with the
sequence of GenBank accession No. NM 001037780 (version NM 001037780.2,
GI:163937848), dog mRNA with the sequence of GenBank accession No. XM_849290
(version XM 849290.3, GI:359323519) and chicken mRNA with the sequence of
GenBank accession No. NM 205251 (version NM 205251.1, GI:46048727).
[0134] In the context of the present invention the terms "detect" or
"detecting"
typically refer to a method that can be used to determine the amount of a
nucleic acid or
a protein or from which such an amount can be inferred. Examples of such
methods
include, but are not limited to, RT-PCR, RNAse protection assay, Northern
analysis,
Western analysis, ELISA, radioimmunoassay or fluorescence titration assay. The
detection method may include an amplification of the signal caused by the
nucleic acid
or protein, such as a polymerase chain reaction (PCR) or the use of the biotin-
streptavidin system, for example in form of a conjugation to an
immunoglobulin, as also
explained in more detail below. The detection method may for example include
the use
of an immunoglobulin, which may be linked to an attached label, such as for
instance in
Western analysis or ELISA. Where desired, an intracellular immunoglobulin may
be
used for detection. Some or all of the steps of detection may be part of an
automated
detection system. Illustrative examples of such systems are automated real-
time PCR
platforms, automated nucleic acid isolation platforms, PCR product analysers
and real-
time detection systems.
[0135] The rate of synthesis of CD62L, CD18 and/or CD45 may also be
assessed by determining the synthesis rate of the respective
protein/polypeptide,
including the post-translational modifications of the initial translation
product. CD62L is
for example synthesized in the form of a pro-L-selectin after removal of the N-
terminal
signal peptide, which directs the protein to its cell membrane location. L-
selectin is then
formed after removal of the N-terminal propeptide. Further, a plurality of N-
linked
glycosylations occur. Likewise, CD162 is for example synthesized in the form
of a pro-
protein after removal of the N-terminal signal peptide. CD162 has complex,
core-2,
sialylated and fucosylated 0-linked oligosaccharides and contains the Sialyl-
Lewisx
(sLex) glycan. Further, CD162 is postranslationally modified by sulfation,
which is
required for P- and L-selectin binding. Any of these synthesis steps may be
detected
alone or in combination, for example based on the accumulation of products of
a post-
translational modification.
[0136] Any method that can be used to detect the presence of a nucleic acid or
a
protein in the context of the present invention. Such a method may include
established
standard procedures well known in the art. Examples of such techniques
include, but are
not limited to, RT-PCR, RNAse protection assay, Northern analysis, Western
analysis,
ELISA, radioimmunoassay or fluorescence titration assay. The detection method
may
include an amplification of the signal caused by the nucleic acid or protein,
such as a
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34
polymerase chain reaction (PCR) or the use of the biotin-streptavidin system,
for
example in form of a conjugation to an immunoglobulin. The detection method
may for
example include the use of an antibody, which may be linked to an attached
label, such
as for instance in Western analysis or ELISA. Where desired, an intracellular
antibody
may be used for detection. Some or all of the steps of detection may be part
of an
automated detection system. Illustrative examples of such a system are
automated real-
time PCR platforms, automated nucleic acid isolation platforms, PCR product
analyzers
and real-time detection systems. The term "antibody" as used herein, is
understood to
include an immunoglobulin and an immunoglobulin fragment that is capable of
specifically binding a selected protein, e.g. L-selectin or a protein specific
for T cells, as
well as a respective proteinaceous binding molecule with immunoglobulin-like
functions.
An antibody may for instance be an EGF-like domain, a Kringle-domain, a
fibronectin
type I domain, a fibronectin type II domain, a fibronectin type III domain, a
PAN
domain, a G1 a domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin
Inhibitor
domain, tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil
(P-type)
domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a
CUB
domain, a thyroglobulin type I repeat, an LDL-receptor class A domain, a Sushi
domain,
a Link domain, a Thrombospondin type I domain, an immunoglobulin domain or a
an
immunoglobulin-like domain (for example a domain antibody or a camel heavy
chain
antibody), a C-type lectin domain, a MAM domain, a von Willebrand factor type
A
domain, a Somatomedin B domain, a WAP-type four disulfide core domain, a F5/8
type
C domain, a Hemopexin domain, an 5H2 domain, an 5H3 domain, a Laminin-type EGF-
like domain, a C2 domain, a "Kappabody" (Ill. et al., Protein Eng (1997) 10,
949-957), a
"Minibody" (Martin et al., EMBO J (1994) 13, 5303-5309), a "Diabody" (Holliger
et al.,
PNAS U.S.A. 90, 6444-6448 (1993)), a "Janusin" (Traunecker et al., EMBO J
(1991) 10,
3655-3659 or Traunecker et al., Int J Cancer (1992) Suppl 7, 51-52), a
nanobody, an
adnectin, a tetranectin, a microbody, an affilin, an affibody or an ankyrin, a
crystallin, a
knottin, ubiquitin, a zinc-finger protein, an autofluorescent protein, an
ankyrin or ankyrin
repeat protein or a leucine-rich repeat protein (cf. also below).
[0137] A measurement of a level or amount may for instance rely on
spectroscopic, photochemical, photometric, fluorometric, radiological,
enzymatic or
thermodynamic means. An example of a spectroscopical detection method is
fluorescence correlation spectroscopy. A photochemical method is for instance
photochemical cross-linking. The use of photoactive, fluorescent, radioactive
or
enzymatic labels, respectively are examples for photometric, fluorometric,
radiological
and enzymatic detection methods. An example of a thermodynamic detection
method is
isothermal titration calorimetry. The measurement used is generally selected
to be of a
sensitivity that allows detection of CD62L or LFA-1 expressing cells in the
range of a
selected threshold value, in particular of a sensitivity that allows
determining whether
CD62L or LFA-1 expressing cells are above or below the threshold value. The
term
"determining" generally refer to any form of measurement, and include
determining if an
element is present or absent. These terms may include either quantitative
and/or
qualitative determinations. Typically a binding partner of CD62L and LFA-1,
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respectively, may be used in combination with a detectable marker. Such a
binding
partner of CD62L or LFA-1 has a detectable affinity and specificity for CD62L
or LFA-
1, respectively. Typically, binding is considered specific when the binding
affinity is
higher than 10-6 M. A binding partner of CD62L and LFA-1, respectively, has in
some
embodiments an affinity of about 10-8 M or higher, or of about 10-9 M or
higher. As
indicated above, in some embodiments T cells in the sample are identified by
the
presence of the CD3 protein on their surface; or T cells may be enriched or
isolated via
the the presence of the CD3 protein on their surface. Identification of CD3 '
T cells may
again be carried out using spectroscopic, photochemical, photometric,
fluorometric,
radiological, enzymatic or thermodynamic means. Identification and enrichment
or
isolation of T cells may likewise be carried out by using a suitable binding
partner of
CD3. Accordingly the above said applies mutatis mutandis to identifying and
enriching
or isolating T cells. Further, T cells may be identified or isolated in a
similar manner,
using suitable surface proteins known in the art, for example the T cell
receptor. In some
embodiments a suitable binding partner of CD3 and a further suitable binding
partner of
a surface protein characteristic for T cells such as the T cell receptor are
combined to
identify CD3 ' T cells. Typically a binding partner of CD3 may be used in
combination
with a detectable marker. Likewise a binding partner of CD3 may be used in
combination
with a detectable marker. In some embodiments a suitable binding partner of
CD3, a
suitable binding partner of a surface protein characteristic for T cells such
as the T cell
receptor and a suitable binding partner of CD62L are combined to identify
CD62L
expressing CD3 ' T cells. In some embodiments a suitable binding partner of
CD3, a
suitable binding partner of a surface protein characteristic for T cells such
as the T cell
receptor and a suitable binding partner of LFA-1 are combined to identify LFA-
1
expressing CD3 ' T cells. In some embodiments a suitable binding partner of
CD3 and a
suitable binding partner of CD62L are combined to identify CD62L expressing T
cells.
In some embodiments a suitable binding partner of CD3 ' and a suitable binding
partner
of LFA-1 are combined to identify LFA-1 expressing T cells.
[0138] The term "specific" as used herein is understood to indicate that the
binding partner is directed against, binds to, or reacts with CD62L and CD3,
respectively. Thus, being directed to, binding to or reacting with includes
that the binding
partner specifically binds to CD62L, LFA-1, CD4, CD8 or CD3, as applicable.
The term
"specifically" in this context means that the binding partner reacts with
CD62L, LFA-1,
CD4, CD8 or CD3, as applicable, or/and a portion thereof, but at least
essentially not
with another protein. The term "another protein" includes any protein,
including proteins
closely related to or being homologous to e.g. CD62L, LFA-1 or CD3 against
which the
binding partner is directed to. The term "does not essentially bind" means
that the
binding partner does not have particular affinity to another protein, i.e.,
shows a cross-
reactivity of less than about 30%, when compared to the affinity to CD62L, LFA-
1 or
CD3. In some embodiments the binding partner shows a cross-reactivity of less
than
about 20%, such as less than about 10%. In some embodiments the binding
partner
shows a cross-reactivity of less than about 9, 8, or 7%, when compared to the
affinity to
CD62L, LFA-1 or CD3. In some embodiments the binding partner shows a cross-
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reactivity of less than about 6%, such as less than about 5%, when compared to
the
affinity to CD62L, LFA-1 or CD3. Whether the binding partner specifically
reacts as
defined herein above can easily be tested, inter alia, by comparing the
reaction of a
respective binding partner with CD62L, with LFA-1 or with CD3, as applicable,
and the
reaction of the binding partner with (an) other protein(s). The term
"specifically
recognizing", which can be used interchangeably with the terms "directed to"
or
"reacting with" means in the context of the present disclosure that a
particular molecule,
generally an immunoglobulin, an immunoglobulin fragment or a proteinaceous
binding
molecule with immunoglobulin-like functions is capable of specifically
interacting with
and/or binding to at least two, including at least three, such as at least
four or even more
amino acids of an epitope as defined herein. Generally the immunoglobulin or
proteinaceous binding molecule can thereby form a complex with the respective
epitope
of e.g. CD62L, LFA-1 or CD3. Such binding may be exemplified by the
specificity of a
"lock-and-key-principle". "Specific binding" can also be determined, for
example, in
accordance with Western blots, ELISA-, RIA-, ECL-, IRMA-tests, FACS, IHC and
peptide scans.
[0139] A respective binding partner of e.g. CD62L, LFA-1 or CD3, as well as a
binding partner for another selected cell-characteristic protein, may be an
immunoglobulin, a fragment thereof or a proteinaceous binding molecule with
immunoglobulin-like functions. Examples of (recombinant) antibody fragments
are
immunoglobulin fragments such as Fab fragments, Fv fragments, single-chain Fv
fragments (scFv), diabodies or domain antibodies (Holt, L.J., et al., Trends
Biotechnol.
(2003), 21, 11, 484-490). An example of a proteinaceous binding molecule with
antibody-like functions is a mutein based on a polypeptide of the lipocalin
family (WO
03/029462, Beste et al., Proc. Natl. Acad. Sci. USA (1999) 96, 1898-1903).
Lipocalins,
such as the bilin binding protein, the human neutrophil gelatinase-associated
lipocalin,
human Apolipoprotein D or glycodelin, possess natural ligand-binding sites
that can be
modified so that they bind to selected small protein regions known as haptens.
Examples
of other proteinaceous binding molecules are the so-called glubodies (see e.g.
international patent application WO 96/23879 or Napolitano, E.W., et al.,
Chemistry &
Biology (1996) 3, 5, 359-367), proteins based on the ankyrin scaffold (Mosavi,
L.K., et
al., Protein Science (2004) 13, 6, 1435-1448) or crystalline scaffold (e.g.
internation
patent application WO 01/04144) the proteins described in Skerra, J. Mol.
Recognit.
(2000) 13, 167-187, AdNectins, tetranectins and avimers. Avimers contain so
called A-
domains that occur as strings of multiple domains in several cell surface
receptors
(Silverman, J., et al., Nature Biotechnology (2005) 23, 1556-1561). Adnectins,
derived
from a domain of human fibronectin, contain three loops that can be engineered
for
immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K., Current
Opinion in
Biotechnology (2006) 17, 653-658). Tetranectins, derived from the respective
human
homotrimeric protein, likewise contain loop regions in a C-type lectin domain
that can be
engineered for desired binding (ibid.). Peptoids, which can act as protein
ligands, are
oligo(N-alkyl) glycines that differ from peptides in that the side chain is
connected to the
amide nitrogen rather than the a carbon atom. Peptoids are typically resistant
to
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37
proteases and other modifying enzymes and can have a much higher cell
permeability
than peptides (see e.g. Kwon, Y.-U., and Kodadek, T., J. Am. Soc. (2007) 129,
1508-
1509). A molecule that forms a complex with a binding partner of e.g. CD62L,
LFA-1 or
CD4 may likewise be an immunoglobulin, a fragment thereof or a proteinaceous
binding
molecule with immunoglobulin-like functions, as explained above. Thus, in an
exemplary embodiment detecting the amount of CD62L, e.g. on a cell surface,
may
carried out using a first antibody or antibody fragment capable of
specifically binding
CD62L, as well as a second antibody or antibody fragment capable of
specifically
binding the first antibody or antibody fragment.
[0140] An immunoglobulin may be monoclonal or polyclonal. The term
"polyclonal" refers to immunoglobulins that are heterogenous populations of
immunoglobulin molecules derived from the sera of animals immunized with an
antigen
or an antigenic functional derivative thereof. For the production of
polyclonal
immunoglobulins, one or more of various host animals may be immunized by
injection
with the antigen. Various adjuvants may be used to increase the immunological
response,
depending on the host species. "Monoclonal immunoglobulins" or "Monoclonal
antibodies" are substantially homogenous populations of immunoglobulins to a
particular
antigen. They may be obtained by any technique which provides for the
production of
immunoglobulin molecules by continuous cell lines in culture. Monoclonal
immuno-
globulins may be obtained by methods well known to those skilled in the art
(see for
example, Kohler et al., Nature (1975) 256, 495-497, and U.S. Patent No.
4,376,110). An
immunoglobulin or immunoglobulin fragment with specific binding affinity only
for e.g.
CD62L, CD3, LFA-1, CD8 or CD4 can be isolated, enriched, or purified from a
prokaryotic or eukaryotic organism. Routine methods known to those skilled in
the art
enable production of both immunoglobulins or immunoglobulin fragments and
proteinaceous binding molecules with immunoglobulin-like functions, in both
prokaryotic and eukaryotic organisms.
[0141] In this regard the terms "immunize", "immunization", or "immunizing"
refer to exposing the immune system of an animal to an antigen or to an
epitope thereof
as illustrated in more detail below. The antigen may be introduced into the
animal using
a desired route of administration, such as injection, inhalation or ingestion.
Upon a
second exposure to the same antigen, the adaptive immune response, in
particular T cell
and B cell responses, is enhanced.
[0142] In more detail, an immunoglobulin may be isolated by comparing its
binding affinity to a protein of interest, e.g. L-selectin, with its binding
affinity to other
polypeptides. Humanized forms of the antibodies of the present invention may
be
generated using one of the procedures known in the art such as chimerization
or CDR
grafting. In general, techniques for preparing monoclonal antibodies and
hybridomas are
well known in the art. Any animal such as a goat, a mouse or a rabbit that is
known to
produce antibodies can be immunized with the selected polypeptide, e.g. L-
selectin.
Methods for immunization are well known in the art. Such methods include
subcutaneous or intraperitoneal injection of the polypeptide. One skilled in
the art will
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38
recognize that the amount of polypeptide used for immunization and the
immunization
regimen will vary based on the animal which is immunized, including the
species of
mammal immunized, its immune status and the body weight of the mammal, as well
as
the antigenicity of the polypeptide and the site of injection.
[0143] The polypeptide may be modified or administered in an adjuvant in order
to increase the peptide antigenicity. Methods of increasing the antigenicity
of a
polypeptide are well known in the art. Such procedures include coupling the
antigen with
a heterologous protein (such as globulin or fl-galactosidase) or through the
inclusion of
an adjuvant during immunization.
[0144] Typically, the immunized mammals are bled and the serum from each
blood sample is assayed for particular antibodies using appropriate screening
assays. As
an illustrative example, anti-CD62L or anti-LFA-1 immunoglobulins may be
identified
by immunoprecipitation of 125I-labeled cell lysates from CD62L or LFA-1-
expressing
cells (see, Sanchez-Madrid et al., 1986 and Hemler et al., 1987). Anti-CD62L
or anti-
LFA-1 immunoglobulins may also be identified by flow cytometry, e.g., by
measuring
fluorescent staining of Ramos cells incubated with an antibody believed to
recognize
CD62L or LFA-1.
[0145] For monoclonal immunoglobulins, lymphocytes, typically splenocytes,
from the immunized animals are removed, fused with an immortal cell line,
typically
myeloma cells, such as 5132/0-Ag14 myeloma cells, and allowed to become
monoclonal
immunoglobulin producing hybridoma cells. Typically, the immortal cell line
such as a
myeloma cell line is derived from the same mammalian species as the
lymphocytes.
Illustrative immortal cell lines are mouse myeloma cell lines that are
sensitive to culture
medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes
using
1500 molecular weight polyethylene glycol ("PEG 1500"). Hybridoma cells
resulting
from the fusion may then be selected using HAT medium, which kills unfused and
unproductively fused myeloma cells (unfused splenocytes die after several days
because
they are not transformed).
[0146] Any one of a number of methods well known in the art can be used to
identify a hybridoma cell which produces an immunoglobulin with the desired
characteristics. Typically the culture supernatants of the hybridoma cells are
screened for
immunoglobulins against the antigen. Suitable methods include, but are not
limited to,
screening the hybridomas with an ELISA assay, Western blot analysis, or
radioimmunoassay (Lutz et al., Exp. Cell Res. [1988] 175, 109-124). Hybridomas
prepared to produce anti-CD62L or anti-LFA-1 immunoglobulins may for instance
be
screened by testing the hybridoma culture supernatant for secreted antibodies
having the
ability to bind to a recombinant CD62L or LFA-1 expressing cell line. To
produce
antibody homologs which are within the scope of the invention, including for
example,
anti-CD62L or anti-LFA-1 antibody homologs, that are intact immunoglobulins,
hybridoma cells that tested positive in such screening assays can be cultured
in a nutrient
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medium under conditions and for a time sufficient to allow the hybridoma cells
to secrete
the monoclonal immunoglobulins into the culture medium. Tissue culture
techniques and
culture media suitable for hybridoma cells are well known in the art. The
conditioned
hybridoma culture supernatant may be collected and for instance the anti-CD62L
immunoglobulins optionally further purified by well-known methods.
Alternatively, the
desired immunoglobulins may be produced by injecting the hybridoma cells into
the
peritoneal cavity of an unimmunized mouse. The hybridoma cells proliferate in
the
peritoneal cavity, secreting the immunoglobulin which accumulates as ascites
fluid. The
immunoglobulin may be harvested by withdrawing the ascites fluid from the
peritoneal
cavity with a syringe.
[0147] Hybridomas secreting the desired immunoglobulins are cloned and the
class and subclass are determined using procedures known in the art. For
polyclonal
immunoglobulins, immunoglobulin containing antisera is isolated from the
immunized
animal and is screened for the presence of immunoglobulins with the desired
specificity
using one of the above-described procedures. The above-described antibodies
may also
be immobilized on a solid support. Examples of such solid supports include
plastics such
as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic
resins
and such as polyacrylamide and latex beads. Techniques for coupling antibodies
to such
solid supports are well known in the art.
[0148] A plurality of conventional display technologies is available to select
an
immunoglobulin, immunoglobulin fragment or proteinaceous binding molecule. Li
et al.
(Organic & Biomolecular Chemistry (2006), 4, 3420-3426) have for example
demonstrated how a single-chain Fv fragment capable of forming a complex with
a
selected DNA adapter can be obtained using phage display. Display techniques
for
instance allow the generation of engineered immunoglobulins and ligands with
high
affinities for a selected target molecule. It is thus also possible to display
an array of
peptides or proteins that differ only slightly, typically by way of genetic
engineering.
Thereby it is possible to screen and subsequently evolve proteins or peptides
in terms of
properties of interaction and biophysical parameters. Iterative rounds of
mutation and
selection can be applied on an in vitro basis.
[0149] In vitro display technology for the selection of peptides and proteins
relies
on a physical linkage between the peptide or protein and a nucleic acid
encoding the same.
A large panel of techniques has been established for this purpose, with the
most commonly
used being phage/virus display, ribosome display, cell-surface display,
'peptides on
plasmids', mRNA display, DNA display, and in vitro compartmentalisation
including
micro-bead display (for reviews see e.g. Rothe, A., et al., FASEB J. (2006)
20, 1599 -1610;
Sergeeva, A., et al., Advanced Drug Delivery Reviews (2006) 58, 1622-1654).
[0150] Different means of physically linking a protein or peptide and a
nucleic
acid are also available. Expression in a cell with a cell surface molecule,
expression as a
fusion polypeptide with a viral/phage coat protein, a stabilised in vitro
complex of an
RNA molecule, the ribosome and the respective polypeptide, covalent coupling
in vitro
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via a puromycin molecule or via micro-beads are examples of ways of linking
the
protein/peptide and the nucleic acid presently used in the art. A further
display technique
relies on a water-in-oil emulsion. The water droplets serve as compartments in
each of
which a single gene is transcribed and translated (Tawfik, D.S., & Griffiths,
A.D., Nature
Biotech. (1998) 16, 652-656, US patent application 2007/0105117). This
physical
linkage between the peptide or protein and the nucleic acid (encoding it)
provides the
possibility of recovering the nucleic acid encoding the selected protein or
peptide.
Compared to techniques such as immunoprecipitation, in display techniques thus
not
only binding partners of a selected target molecule can be identified or
selected, but the
nucleic acid of this binding partner can be recovered and used for further
processing.
Present display techniques thus provide means for e.g. target discovery, lead
discovery and lead optimisation. Vast libraries of peptides or proteins, e.g.
antibodies, potentially can be screened on a large scale.
[0151] As indicated above, a detectable marker may be coupled to a binding
partner of CD62L, of LFA-1, of CD4, of CD8 or CD3, as the case may be, or a
molecule
that forms a complex with the binding partner of CD62L, LFA-1, CD4, CD8 or
CD3. A
respective detectable marker, which may be coupled to a binding partner of
CD62L,
LFA-1, CD4, CD8 or CD3, or a molecule that forms a complex therewith, may be
an
optically detectable label, a fluorophore, or a chromophore. Examples of
suitable labels
include, but are not limited to, an organic molecule, an enzyme, a
radioactive,
fluorescent, and/or chromogenic moiety, a luminescent moiety, a hapten,
digoxigenin,
biotin, a metal complex, a metal and colloidal gold. Accordingly an excitable
fluorescent
dye, a radioactive amino acid, a fluorescent protein or an enzyme may for
instance be
used to detect e.g. the level of CD62L. Examples of suitable fluorescent dyes
include, but
are not limited to, fluorescein isothiocyanate, 5,6-carboxymethyl fluorescein,
Cascade
Blue , Oregon Green , Texas red, nitrobenz-2-oxa-1,3-diazol-4-yl, coumarin,
dansyl
chloride, rhodamine, amino-methyl coumarin, DAPI, Eosin, Erythrosin, BODIPYO,
pyrene, lissamine, xanthene, acridine, an oxazine, phycoerythrin, a Cy dye
such as Cy3,
Cy3.5, Cy5, Cy5PE, Cy5.5, Cy7, Cy7PE or Cy7APC, an Alexa dye such as Alexa
647,
and NBD (Naphthol basic dye). Examples of suitable fluorescent protein
include, but are
not limited to, EGFP, emerald, EYFP, a phycobiliprotein such as phycoerythrin
(PE) or
allophycocyanin, Monomeric Red Fluorescent Protein (mRFP), mOrange, mPlum and
mCherry. In some embodiments a reversibly photoswitchable fluorescent protein
such as
Dronpa, bsDronpa and Padron may be employed (Andresen, M., et al., Nature
Biotechnology (2008) 26, 9, 1035). Regarding suitable enzymes, alkaline
phosphatase,
soybean peroxidase, or horseradish peroxidase may serve as a few illustrative
examples.
In some embodiments a method of detection may include electrophoresis, HPLC,
flow
cytometry, fluorescence correlation spectroscopy or a modified form of these
techniques.
Some or all of these steps may be part of an automated separation/detection
system.
[0152] In some embodiments the binding partner of e.g. CD62L, LFA-1 or CD3,
as well as a binding partner for another selected cell-characteristic protein,
further
includes a capture molecule. Such a capture molecule allows immobilization of
the
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binding partner, and thereby also of a complex formed between e.g. CD62L, LFA-
1 or
CD3, or another selected cell-characteristic protein, on a surface or on a
polymeric
molecule, including an immunoglobulin, an immunoglobulin fragment or a
proteinaceous
binding molecule with antibody-like functions. A respective surface may for
instance be
the surface of a micro- or nanoparticle, the surface of a container or the
surface of a
particularly designed device used for presentation purposes during
measurement. A
micro- or nanoparticle may in some embodiments include, essentially consist of
or
consist of a metal, a metalloid or a polymer. In some embodiments the micro-
or
nanoparticle is magnetic, such as paramagnetic or supermagnetic. The capture
molecule
may be immobilised on the surface via a covalent bond or a non-covalent bond.
[0153] The capture molecule has an affinity to a binding partner of the
capture
molecule and is capable of forming a complex with the binding partner of the
capture
molecule. Hence, the capture molecule and the binding partner of the capture
molecule
define a specific binding pair. Accordingly, a pair of capture molecule and
binding
partner of the capture molecule may be selected as desired, for example
according to the
binding partner of CD62L, LFA-1 or CD3 or to the measurement conditions used
in
detection of for instance CD62L. Examples of a capture molecule include, but
are not
limited to, a nucleic acid molecule, an oligonucleotide, a protein, an
oligopeptide, a
polysaccharide, an oligosaccharide, a synthetic polymer, a drug candidate
molecule, a
drug molecule, a drug metabolite, a metal ion, and a vitamin. Three
illustrative examples
of suitable capture molecule are biotin, dinitrophenol or digoxigenin. Where
the binding
partner of the capture molecule is a protein, a polypeptide, or a peptide,
further examples
of a capture molecule include, but are not limited to, a streptavidin binding
tag such as
the STREP-TAGS described in US patent application US 2003/0083474, US patent
5,506,121 or 6,103,493, an immunoglobulin domain, maltose-binding protein,
glutathione-S-transferase (GST), calmodulin binding peptide (CBP), FLAG-
peptide (e.g.
of the sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Gly), the T7 epitope (Ala-Ser-
Met-
Thr-Gly-Gly-Gln-Gln-Met-Gly), maltose binding protein (MBP), the HSV epitope
of the
sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of herpes simplex virus
glycoprotein D, the Vesicular Stomatitis Virus Glycoprotein (VSV-G) epitope of
the
sequence Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys, the hemagglutinin (HA)
epitope of the sequence Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala and the "myc"
epitope of
the transcription factor c-myc of the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-
Asp-
Leu. Where the binding partner of the capture molecule is a nucleic acid, a
polynucleotide or an oligonucleotide, a capture molecule may furthermore be an
oligonucleotide. Such an oligonucleotide tag may for instance be used to
hybridize to an
immobilised oligonucleotide with a complementary sequence.
[0154] As an illustrative example, the capture molecule may be a metal ion
bound by a respective metal chelator, such as ethylenediamine,
ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid
(EGTA),
diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also
called
nitrilotriacetic acid, NTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-
tetraacetic acid
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(BAPTA), 2,3-dimercapto- 1 -propanol (dimmercaprol), porphine or heme. A
respective
metal ion may define a receptor molecule for a peptide of a defined sequence,
which may
also be included in a protein. In line with the standard method of immobilised
metal
affinity chromatography used in the art, for example an oligohistidine tag of
a respective
peptide or protein is capable of forming a complex with copper (Cu2'), nickel
(Ni2),
cobalt (Co2), or zink (Zn2) ions, which can for instance be presented by means
of the
chelator nitrilotriacetic acid (NTA).
[0155] The capture molecule may be immobilised on a surface (vide infra) such
as the surface of a particle such as a metal containing bead. The capture
molecule may be
immobilised by any means. It may be immobilised on a portion or the entire
area of a
surface. An illustrative example is the mechanical spotting of a nucleic acid
capture
molecule onto a metal surface. This spotting may be carried out manually, e.g.
by means
of a pipette, or automatically, e.g. by means of a micro robot. As an
illustrative example,
a protein capture molecule, a peptide capture molecule or the polypeptide
backbone of a
PNA capture molecule may be covalently linked to a gold surface via a thio-
ether-bond.
[0156] In embodiments where both the capture molecule and the binding partner
of the capture molecule are a nucleic acid molecule, including an
oligonucleotide, the
capture molecule typically has a nucleotide sequence that is at least
partially
complementary to a portion of a strand of the binding partner of the capture
molecule. As
a further illustrative example, Avidin or streptavidin may be employed to
immobilise a
biotinylated nucleic acid, or a biotin containing monolayer of gold may be
employed
(Shumaker-Parry, J.S., et al., Anal. Chem. (2004) 76, 918). As another
illustrative
example, the capture molecule may be a metal ion bound by a respective metal
chelator
(see above).
[0157] In this regard the term "capture probe" as used herein refers to
matter,
such as a molecule, in particular a polymeric molecule, that can bind a
nucleic acid
molecule such as a DNA or an RNA molecule, including an mRNA molecule, as well
as
a peptide, a protein, a saccharide, a polysaccharide or a lipid through an
interaction that
is sufficient to permit the agent to form a complex with the nucleic acid
molecule,
peptide, protein or saccharide, a polysaccharide or a lipid, generally via non-
covalent
bonding. In some embodiments the capture probe is a PNA molecule. As indicated
above, a PNA molecule is a nucleic acid molecule in which the backbone is a
pseudopeptide rather than a sugar. Accordingly, PNA generally has a charge
neutral
backbone, in contrast to DNA or RNA. Nevertheless, PNA is capable of
hybridising at
least complementary and substantially complementary nucleic acid strands, just
as e.g.
DNA or RNA (to which PNA is considered a structural mimic). In some
embodiments
the capture probe is an aptamer, including a Spiegelmer0, described in e.g. WO
01/92655. An aptamer is typically a nucleic acid molecule that can be selected
from a
random nucleic acid pool based on its ability to bind a selected other
molecule such as a
peptide, a protein, a nucleic acid molecule a or a cell. Aptamers, including
Spiegelmers,
are able to bind molecules such as peptides, proteins and low molecular weight
compounds. Spiegelmers0 are composed of L-isomers of natural oligonucleotides.
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Aptamers are engineered through repeated rounds of in vitro selection or
through the
SELEX (systematic evolution of ligands by exponential enrichment) technology.
The
affinity of Spiegelmers to their target molecules often lies in the pico- to
nanomolar
range and is thus comparable to immunoglobulins. An aptamer may also be a
peptide. A
peptide aptamer consists of a short variable peptide domain, attached at both
ends to a
protein scaffold.
[0158] In typical embodiments the capture probe is an immunoglobulin or of a
proteinaceous binding molecule with immunoglobulin-like functions as defined
above. In
some embodiments the capture probe may be detectably labelled as explained
above, for
example where the capture probe is intended to be used together with a
detection agent
that binds to the biomarker and/or the capture probe. The capture probe and/or
a
respective detection agent may be detectably labelled by linking the same,
typically
covalently, to a detectable marker such as a radioactive label, a fluorescent
moiety, a
chemical entity of low molecular weight, an oligonucleotide, an enzyme, or a
protein
such as a fluorescent protein such as a Green Fluorescent Protein (cf. above).
It is
understood that the method may also include any molecules which can be used to
indirectly indicate the level of the target molecule of interest such as
CD62L, CD3, CD4,
CD8, CD18 or CD11a. The capture probe may in some embodiments be an
immunoglobulin, a portion thereof, a proteinaceous binding molecule with
immunoglobulin-like functions, a receptor for the biomarker or a portion
thereof or a
ligand for the biomarker or a portion thereof. The detection agent may in some
embodiments be an immunoglobulin, a portion thereof, a proteinaceous binding
molecule
with immunoglobulin-like functions, a receptor for the biomarker or a portion
thereof, a
ligand for the biomarker or a portion thereof or a capture probe or a portion
thereof.
[0159] In some embodiments a capture probe capable of binding a particular
target nucleic acid molecule such as an mRNA molecule encoding e.g. CD62L,
CD18 or
CD1 la, is a nucleic acid molecule that includes a nucleotide sequence that is
at least
partially complementary to a portion of a strand of such a target nucleic acid
molecule. A
nucleotide sequence is the complement of another nucleotide sequence if all of
the
nucleotides of the first sequence are complementary to all of the nucleotides
of the
second sequence. Accordingly, the respective nucleotide sequence will
specifically
hybridise to, or undergo duplex formation with, the respective portion of the
target
nucleic acid molecule under suitable hybridisation assay conditions, in
particular of ionic
strength and temperature.
[0160] As an illustrative example, a single-stranded nucleic acid molecule may
be selected as a nucleic acid capture probe. Such a single-stranded nucleic
acid molecule
may have a nucleic acid sequence that is at least partially complementary to
at least a
portion of a strand of the target nucleic acid molecule. The respective
nucleotide
sequence of the nucleic acid capture molecule may for example be 70, for
example 80 or
85, including 100 % identical to another nucleic acid sequence. The higher the
percentage to which the two sequences are complementary to each other (i.e.
the lower
the number of mismatches), the higher is typically the sensitivity of the
method of the
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invention. In typical embodiments the respective nucleotide sequence is
substantially
complementary to at least a portion of the target nucleic acid molecule.
"Substantially
complementary" as used herein refers to the fact that a given nucleic acid
sequence is at
least 90 % identical to another nucleic acid sequence. A substantially
complementary
nucleic acid sequence is in some embodiments 95 %, such as 100 % identical to
another
nucleic acid sequence. The term "complementary" or "complement" refers to two
nucleotides that can form multiple favourable interactions with one another.
Such
favourable interactions are specific association between opposing or adjacent
pairs of
nucleic acid (including nucleic acid analogue) strands via matched bases, and
include
Watson-Crick base pairing. As an illustrative example, in two given nucleic
acid
molecules (e.g. DNA molecules) the base adenosine is complementary to thymine
or
uracil, while the base cytosine is complementary to guanine. A nucleic acid
probe used in
the context of the present invention may be used to probe the sample by usual
hybridization methods to detect the presence of nucleic acid molecules
encoding e.g.
CD62L, CD18 or CD1 la.
[0161] The term "nucleic acid molecule" as used herein refers to any nucleic
acid in any possible configuration, such as single stranded, double stranded
or a
combination thereof. Examples of nucleic acids include for instance DNA
molecules,
RNA molecules, analogues of the DNA or RNA generated using nucleotide
analogues or
using nucleic acid chemistry, locked nucleic acid molecules (LNA), protein
nucleic acids
molecules (PNA), alkylphosphonate and alkylphosphotriester nucleic acid
molecules and
tecto-RNA molecules (e.g. Liu, B., et al., J. Am. Chem. Soc. (2004) 126, 4076-
4077).
LNA has a modified RNA backbone with a methylene bridge between C4' and 02',
providing the respective molecule with a higher duplex stability and nuclease
resistance.
Alkylphosphonate and alkylphosphotriester nucleic acid molecules can be viewed
as a
DNA or an RNA molecule, in which phosphate groups of the nucleic acid backbone
are
neutralized by exchanging the P-OH groups of the phosphate groups in the
nucleic acid
backbone to an alkyl and to an alkoxy group, respectively. DNA or RNA may be
of
genomic or synthetic origin and may be single or double stranded. Such nucleic
acid can
be e.g. mRNA, cRNA, synthetic RNA, genomic DNA, cDNA synthetic DNA, a
copolymer of DNA and RNA, oligonucleotides, etc. A respective nucleic acid may
furthermore contain non-natural nucleotide analogues and/or be linked to an
affinity tag
or a label.
[0162] Many nucleotide analogues are known and can be used in nucleic acids
used in the methods of the invention. A nucleotide analogue is a nucleotide
containing a
modification at for instance the base, sugar, or phosphate moieties. As an
illustrative
example, a substitution of 2'-OH residues of siRNA with 2'F, 2'0-Me or 2'H
residues is
known to improve the in vivo stability of the respective RNA. Modifications at
the base
moiety may be a natural or a synthetic modification of A, C, G, and T/U, a
different
purine or pyrimidine base, such as uracil-5-yl, hypoxanthin-9-yl, and 2-
aminoadenin-9-
yl, as well as a non-purine or a non-pyrimidine nucleotide base. Other
nucleotide
analogues serve as universal bases. Examples of universal bases include 3-
nitropyrrole
and 5-nitroindole. Universal bases are able to form a base pair with any other
base. Base
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modifications often can be combined with for example a sugar modification,
such as for
instance 2'-0-methoxyethyl, e.g. to achieve unique properties such as
increased duplex
stability.
[0163] Interactions between two or more nucleic acid molecules are generally
sequence driven interactions referred to as hybridization. Sequence driven
interaction is
an interaction that occurs between two nucleotides or nucleotide analogs or
nucleotide
derivatives in a nucleotide specific manner (supra). Typically sequence driven
interactions occur on the Watson-Crick face or Hoogsteen face of the
respective
nucleotide. The hybridization of two nucleic acids is affected by a number of
conditions
and parameters known to those skilled in the art. For example, the salt
concentrations,
pH, and temperature of the reaction all affect whether two nucleic acid
molecules will
hybridize. For example, in some embodiments selective hybridization conditions
can be
defined as stringent hybridization conditions. For example, stringency of
hybridization is
controlled by both temperature and salt concentration of either or both of the
hybridization and washing steps. For example, conditions of hybridization that
achieve
selective interactions between complementary sequences may involve
hybridization in
high ionic strength solution (6 x SSC or 6 x SSPE) at a temperature that is in
the range
from about 12 to about 25 C below the Tm, the melting temperature at which
half of the
molecules dissociate from their hybridization partners, followed by washing at
a
combination of temperature and salt concentration chosen so that the washing
temperature is in the range from about 5 C to about 20 C below the Tm. The
temperature and salt conditions are readily determined empirically in
preliminary
experiments in which samples of reference DNA immobilized on filters are
hybridized to
a labelled nucleic acid of interest and then washed under conditions of
different
stringencies. Hybridization temperatures are typically higher for DNA-RNA and
RNA-
RNA hybridizations than for DNA-DNA hybridizations.
[0164] In order to obtain nucleic acid probes having nucleotide sequences
which
correspond to altered portions of the amino acid sequence of the polypeptide
of interest,
chemical synthesis can be carried out. The synthesized nucleic acid probes may
be first
used as primers in a polymerase chain reaction (PCR) carried out in accordance
with
recognized PCR techniques, essentially according to standard PCR protocols
utilizing the
appropriate template, in order to obtain the probes that can be used in the
context of the
present invention.
[0165] One skilled in the art will readily be able to design such probes based
on
a sequence as referred to herein using methods of computer alignment and
sequence
analysis well known in the art. As explained above, a respective hybridization
probe can
be labelled by standard labelling techniques using a detectable marker, such
as with a
radiolabel, enzyme label, fluorescent label, biotin-avidin label, or
chemiluminescence
(supra). After hybridization, the probes may be visualized using known
methods. A
nucleic acid probe may be immobilized on a solid support. Examples of such
solid
supports include, but are not limited to, plastics such as polycarbonate,
complex
carbohydrates such as agarose and sepharose, and acrylic resins, such as
polyacrylamide
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and latex beads. As an illustrative example one or more nucleic acid probes
may be
bound to or immobilized on a solid support. The solid support may be a chip,
for
example a DNA microchip. Techniques for coupling nucleic acid probes to such
solid
supports are well known in the art.
[0166] The most frequently used methods for determining the concentration of
nucleic acids include the detection by autoradiography, fluorescence,
chemiluminescence
or bioluminescence as well as electrochemical and electrical techniques. A
further
suitable technique is the electrical detection of a target nucleic acid
molecule as disclosed
in international patent applications WO 2009/041917 and WO 2008/097190, both
being
incorporated herein by reference in their entirety. A technique for the
specific detection
of a selected nucleic acid well established in the art is based on the
hybridisation between
a nucleic acid capture probe and a target nucleic acid. Typically the
respective nucleic
acid capture probe is immobilised onto a solid support, and subsequently one
of the
above mentioned detection methods is employed.
[0167] As indicated above, an immunoglobulin labeled with a fluorescence dye
may for instance be used to optically detect the presence of a certain protein
or
polypeptide. Nucleic acid intercalating dyes, such as YOYO, JOJO, BOBO, POPO,
TOTO, LOLO, SYBR, SYTO, SYTOX, PicoGreen, or Oligreen as available from
Molecular Probes, may be used for optical detection.
[0168] In some embodiments determining the level of expression of the gene of
interest includes determining the level of transcription into mRNA. RNA
encoding the
protein of interest in the sample, such as CD62L, CD11A, CD18, CD3, CD4 or CD8
may
be amplified using any available amplification technique, such as polymerase
chain
reaction (PCR), including multiplex PCR, nested PCR and amplification
refractory
mutation specific (ARMS) PCR (also called allele-specific PCR (AS-PCR),
rolling circle
amplification (RCA), nucleic acid sequence based amplification (NASBA), ligase
chain
reaction (LCR), QB replicase chain reaction, loop-mediated isothermal
amplification
(LAMP), transcription mediated amplification (TMA) and strand displacement
amplification (SDA), including genome strand displacement amplification
(WGSDA),
multiple strand displacement amplification (MSDA), and gene specific strand
displacement amplification (GS-MSDA). Detection of the obtained amplification
products may be performed in numerous ways known in the art. Examples include,
but
are not limited to, electrophoretic methods such as agarose gel
electrophoresis in
combination with a staining such as ethidium bromide staining. In other
embodiments the
method of the invention is accompanied by real time detection, such as real
time PCR. In
these embodiments the time course of the amplification process is monitored. A
means of
real time detection commonly used in the art involves the addition of a dye
before the
amplification process. An example of such a dye is the fluorescence dye SYBR
Green,
which emits a fluorescence signal only when bound to double-stranded nucleic
acids.
[0169] As explained above, typically a detectable label or marker is used.
Such a
marker or label may be included in a nucleic acid that includes the sequence
to be
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amplified. A marker may also be included in a primer or a probe. It may also
be
incorporated into the amplification product in the course of the reaction. In
some
embodiments such a marker compound, e.g. included in a nucleic acid, is an
optically
detectable label, a fluorophore, or a chromophore. An illustrative example of
a marker
compound is 6-carboxyfluorescein (FAM).
[0170] As an illustrative example, real-time PCR may be used to determine the
level of RNA encoding the protein of interest in the sample, such as CD62L,
CD11A,
CD18, CD3, CD4 or CD8. Such a PCR procedure is carried out under real time
detection, so that the time course of the amplification process is monitored.
PCR is
characterised by a logarithmic amplification of the target sequences. For the
amplification of RNA, a reverse transcriptase-PCR is used. Design of the
primers and
probes required to detect expression of a biomarker of the invention is within
the skill of
a practitioner of ordinary skill in the art. In some embodiments RNA from the
sample is
isolated under RNAse free conditions and then converted to DNA via the use of
a reverse
transcriptase. Reverse transcription may be performed prior to RT-PCR analysis
or
simultaneously, within a single reaction vessel. RT-PCR probes are
oligonucleotides that
have a fluorescent moiety, also called reporter dye, attached to the 5' end
and a quencher
moiety coupled to the 3' end (or vice versa). These probes are typically
designed to
hybridize to an internal region of a PCR product. In the unhybridized state,
the proximity
of the fluor and the quench molecules prevents the detection of fluorescent
signal from
the probe. During PCR amplification, when the polymerase replicates a template
on
which an RT-PCR probe is bound, the 5'-3' nuclease activity of the polymerase
cleaves
the probe. Thereby the fluorescent and quenching moieties are decoupled.
Fluorescence
increases then in each cycle, in a manner proportional to the amount of probe
cleavage.
Fluorescence signal emitted from the reaction can be measured or followed over
time
using equipment which is commercially available using routine and conventional
techniques. Quantitation of biomarker RNA in a sample being evaluated may be
performed by comparison of the amplification signal to that of one or more
standard
curves where known quantities of RNA were evaluated in a similar manner. In
some
embodiments, the difference in biomarker expression is measured as the
difference in
PCR cycle time to reach a threshold fluorescence, or
[0171] In some embodiments the level or amount of CD62L, LFA-1 and/or CD3
on the surface of cells in the sample is determined using a flow cytometry
based
analysis, typically in combination with immunofluorescence. Immunofluorescence
is
generally achieved using a binding partner as described above, which is linked
to, or
includes, a fluorophore as a detectable marker (supra). Flow cytometry is a
technique for
counting, examining, and sorting microscopic particles such as biological
cells
suspended in a stream of fluid. It allows a simultaneous multiparametric
analysis of the
physical and chemical characteristics of single cells flowing through an
optical or
electronic detection device. An illustrative example of a well established
flow cytometry
based analysis in the art is fluorescence-activated cell sorting (FACS). FACS
allows
sorting a heterogeneous mixture of cells into a plurality of containers, one
cell at a time,
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based upon the specific light scattering and fluorescent characteristics of
each cell.
Thereby FACS allows the sorting of subpopulations of cells of interest and
their further
use in in vitro and in vivo assays. FACS is often be used in combination with
monoclonal
antibodies as a reagent to detect cells as having a particular antigen,
indicative of an
expressed protein (supra).
[0172] This technique allows the concurrent fast, objective and quantitative
recording of fluorescent signals from individual cells and the physical
separation of
respective cells according to particular interest. Fluorescent signals used in
flow
cytometry, for instance when quantifying and/or sorting cells by any marker
present on
or in the cell, are typically fluorescently-tagged antibody preparations or
fluorescently-
tagged ligands for binding to antibodies or other antigen-, epitope- or ligand-
specific
agent, such as with biotin/avidin binding systems or fluorescently-labelled
and optionally
addressable beads (e.g. LUMINEXO microspheres). Depending of the equipment
used,
any desired detectable marker or combination of detectable markers can be
detected by
the optics and/or electronics of a flow cytometer. Current three-laser,
"multidimensional", FACS machines enable up to 14 simultaneous single-cell
measurements, such as 2 light scatter detectors and 12 fluorescence plus
forward
detectors allowing for example the detection of fluorescent
surface/intracellular markers.
As an illustrative example, the three lasers of a FACS machine may be a
krypton laser
operating at 407 nm, an argon laser operating at 488 nm, and a dye laser
operating at 595
11111.
[0173] It is to be understood that expression to be detected is not limited to
the
biomarkers as such, but also that of proteins or mRNA which regulates the
biomarker
(such as transcription factors), metabolites of said biomarker, or any subunit
thereof, or
any molecules whose expression can be correlated with the biomarkers. For
example, the
detection of LFA-1 expression can also include detecting the protein or mRNA
of CD1la
and Runx3.
[0174] For CD62L, monitoring may be targeted at soluble CD62L in the patient.
In this case, the sample to be tested may be any bodily fluids such as serum
or CSF.
[0175] The FACS technique has been used extensively in relation to antigens
expressed on the surface of cells, including cells that remain alive during,
and after,
FACS. Similarly, the method has been used with intracellular reporter gene
systems
based on the expression of a detectably labelled gene product by the cell.
Accordingly,
the technique not only allows detecting the presence of e.g. CD62L, LFA-1, CD4
or CD8
on the cell surface, but also detecting the presence of RNA or DNA within the
cell, for
example RNA encoding CD62L and CD3 or CD4 (vide infra). Therefore FACS can
also
be used to determine the amount of nucleic acid formation from the SELL gene,
which
encodes CD62L, in cells, such as T cells, including CD4 T cells or CD8' T
cells, of the
sample from the subject.
[0176] In some embodiments determining the amount of CD62L, LFA-1, CD3,
CD4 and/or CD8 on the surface of cells in the sample is carried out by
determining the
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amount of CD62L, LFA-1, CD4 and/or CD8 that is accessible in the sample. Such
a
method can be taken to be a method of determining extracellular CD62L, LFA-1,
CD3,
CD4 and/or CD8 in the sample. In embodiments where cells such as T cells are
immobilized on a surface, for example using a capture reagent as detailed
above, before
determining the amount of e.g. CD62L, LFA-1 and/or CD3, any soluble LFA-1,
CD62L
and/or CD3 can easily be removed, for example by way of washing. In such
embodiments therefore only CD62L, LFA-1 and/or CD3 on the surface of cells is
being
determined. An illustrative example of a suitable technique in this regard is
a radiolabel
assay such as a Radioimmunoassay (RIA) or an enzyme-immunoassay such as an
Enzyme Linked Immunoabsorbent Assay (ELISA). While a RIA is based on the
measurement of radioactivity associated with a complex formed between an
immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like
functions and an antigen, an ELISA is based on the measurement of an enzymatic
reaction associated with a complex formed between an immunoglobulin or a
proteinaceous binding molecule with immuneglobulin-like functions and an
antigen.
Typically a radiolabel assay or an enzyme-immunoassay involves one or more
separation
steps in which a binding partner of e.g. CD62L, LFA-1 or CD3 that has not
formed a
complex with CD62L, LFA-1 or CD3 is being removed, thereby leaving only
binding
partner of CD62L, LFA-1 or CD3 behind, which has formed a complex with CD62L,
LFA-1 or CD3. This allows the generation of specific signals originating from
the
presence of CD62L, LFA-1 or CD3.
[0177] An ELISA or RIA test can be competitive for measuring the amount of
CD62L, LFA-1, CD3, CD4 and/or CD8, i.e. the amount of antigen. For example, an
enzyme labelled antigen is mixed with a test sample containing antigen, which
competes
for a limited amount of immunoglobulin or a proteinaceous binding molecule
with
immunoglobulin-like functions. The reacted (bound) antigen is then separated
from the
free material, and its enzyme activity is estimated by addition of substrate.
An alternative
method for antigen measurement is the double immunoglobulin/proteinaceous
binding
molecule sandwich technique. In this modification a solid phase is coated with
specific
immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like
functions. This is then reacted with the sample from the subject that contains
the antigen.
Then enzyme labelled specific immunoglobulin/proteinaceous binding molecule is
added, followed by the enzyme substrate. The 'antigen' in the test sample is
thereby
'captured' and immobilized on to the sensitized solid phase where it can
itself then
immobilize the enzyme labelled immunoglobulin/proteinaceous binding molecule.
This
technique is analogous to the immunoradiometric assays.
[0178] In an indirect ELISA method, an antigen is immobilized by passive
adsorption on to the solid phase. A test serum may then be incubated with the
solid phase
and any immunoglobulin in the test serum forms a complex with the antigen on
the solid
phase. Similarly a solution of a proteinaceous binding molecule with
immunoglobulin-
like functions may be incubated with the solid phase to allow the formation of
a complex
between the antigen on the solid phase and the proteinaceous binding molecule.
After
washing to remove unreacted serum components an anti-immunoglobulin
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immunoglobulin anti-proteinaceous binding molecule immunoglobulin, linked to
an
enzyme is contacted with the solid phase and incubated. Where the second
reagent is
selected to be a proteinaceous binding molecule with immunoglobulin-like
functions, a
respective proteinaceous binding molecule that specifically binds to the
proteinaceous
binding molecule or the immunoglobulin directed against the antigen is used. A
complex
of the second proteinaceous binding molecule or immunoglobulin and the first
proteinaceous binding molecule or immunoglobulin, bound to the antigen, is
formed.
Washing again removes unreacted material. In the case of RIA radioactivity
signals are
being detected. In the case of ELISA the enzyme substrate is added. Its colour
change
will be a measure of the amount of the immobilized complex involving the
antigen,
which is proportional to the antibody level in the test sample.
[0179] In another embodiment the immunoglobulin or the proteinaceous binding
molecule with immunoglobulin-like functions may be immobilized onto a surface,
such
as the surface of a polymer bead (supra), or coated onto the surface of a
device such as a
polymer plate or a glass plate. As a result the immune complexes can easily be
separated
from other components present by simply washing the surface, e.g. the beads or
plate.
This is the most common method currently used in the art and is referred to as
solid
phase RIA or ELISA. This embodiment may be particularly useful for determining
the
amount of CD62L, LFA-1, CD4 and/or CD8 on the surface of cells (cf. also
above). On a
general basis, in any embodiment of a radiolabel assay or of an enzyme-
immunoassay
passive adsorption to the solid phase can be used in the first step.
Adsorption of other
reagents can be prevented by inclusion of wetting agents in all the subsequent
washing
and incubation steps. It may be advantageous to perform washing to prevent
carry-over
of reagents from one step to the next.
[0180] Various other modifications of ELISA have been used in the art. For
example, a system where the second proteinaceous binding molecule or
immunoglobulin
used in the double antibody sandwich method is from a different species, and
this is then
reacted with an anti-immunoglobulin enzyme conjugate or an anti-proteinaceous
binding
molecule enzyme conjugate. This technique comes with the potential advantage
that it
avoids the labelling of the specific immunoglobulin or proteinaceous binding
molecule,
which may be in short supply and of low potency. This same technique can be
used to
assay immunoglobulin or proteinaceous binding molecule where only an impure
antigen
is available; the specific reactive antigens are selected by the antibody
immobilized on
the solid phase.
[0181] In another example of an ELISA assay for an antigen, a surface, a
specific antigen is immobilized on a surface, e.g. a plate used, and the
surface is then
incubated with a mixture of reference immunoglobulins or proteinaceous binding
molecules and a test sample. If there is no antigen in the test sample the
reference
immunoglobulin or proteinaceous binding molecule becomes fixed to an antigen
sensitized surface. If there is antigen in the test solution this combines
with the reference
immunoglobulin or proteinaceous binding molecule, which cannot then react with
the
sensitized solid phase. The amount of immunoglobulin/proteinaceous binding
molecule
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attached is then indicated by an enzyme labelled anti-globulin/anti- binding
molecule
conjugate and enzyme substrate. The amount of inhibition of substrate
degradation in the
test sample (as compared with the reference system) is proportional to the
amount of
antigen in the test system.
[0182] Yet a further technique that can also be carried out to quantify and
thus
determine the amount of CD62L, LFA-1, CD3, CD4 and/or CD8 on the surface of
cells
in the sample is Fluorescence Microscopy, including Ratio Fluorescence
Microscopy.
Fluorescence microscopy has long been used as a descriptive adjunct to
quantitative
biochemical techniques in studies of cellular organization and physiology. In
the late
1970s, sensitive imaging detectors became commercially available and gave
fluorescence
microscopy the potential to be a quantitative tool. However, because of the
prohibitive
cost and sophistication of high-speed image processing computers, quantitative
fluorescence microscopy was generally limited to relatively few laboratories
with a
specific interest in "digital imaging microscopy." This situation has changed
in the past
years with the revolution in digital technology. Inexpensive personal
computers are
now capable of tasks that once required large mainframe computers.
[0183] Integrated optical imaging systems are commercially available that are
capable of processing an entire assay from the biological preparation to the
final data. In
parallel, significant improvements have been made in optical elements and
imaging
hardware. Sensitive fluorescent indicators of a variety of physiologically
important
properties have been introduced, and new fluorescent reagents are continually
being
developed for sensitively and specifically characterizing the intracellular
distribution of
proteins, nucleotides, ions, and lipids.
[0184] As quantitative microscopy becomes more widely available, researchers
new to fluorescence microscopy should be aware of the factors that may
complicate
quantification of fluorescence. The amount of fluorescence detected is
affected by the
properties of illumination sources, the optical and spectroscopic properties
of the
microscope, and the resolution, sensitivity, and signal-to-noise properties of
the detector.
Fluorescence emissions are attenuated by the photobleaching that accompanies
illumination. At high concentrations of fluorophore, interactions between
fluorophore
moieties can alter the amount and/or spectrum of fluorescence emissions. For
certain
fluorophores, fluorescence is also sensitive to the immediate physical
environment (i.e.,
for example, ionic composition) of the fluorophore.
[0185] In ratio fluorescence microscopy two fluorescence images are collected
and the parameter of interest is quantified as a ratio of the fluorescence in
one image to
that in the other image. An illustrative example of a ratio fluorescent ion
indicator
includes, but is not limited to, fluorescein and fura-2, the excitation
spectra of which
change shape upon binding protons or calcium ions, respectively. In the case
of
fluorescein, fluorescence excited by 490 run light is efficiently quenched by
proton
binding, whereas fluorescence excited by 450 nm light is relatively
unaffected. Although
the quantity of fluorescein fluorescence emitted by a volume when excited with
490 nm
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light depends on the pH of that volume, it is also affected by other factors,
including the
concentration of fluorescein in the volume. However, the ratio of fluorescence
excited by
90 nm light to that excited by 450 nm depends on pH, but is relatively
independent of
many variables that affect quantification in single wavelength images:
fluorophore
concentration, photobleaching, lateral heterogeneity in illumination and
detector
sensitivity, and differences in optical path length. Spectroscopic variation
in illumination
and detection is circumvented by calibrating the microscopic system with known
pH
standards.
[0186] Fluorescence ratio images may be collected by sequentially exciting the
sample with two different wavelengths of light and sequentially collecting two
different
images, by exciting the sample with a single wavelength of light and
collecting images
formed from light of two different emission wavelengths, or by exciting the
sample with
two wavelengths and collecting emissions of two wavelengths. Ion indicators
have been
developed for both excitation ratio microscopy (i.e., for example, fura-2 for
calcium and
fluorescein for pH) and for emission ratio microscopy (i.e., for example, indo-
1 for
calcium and SNARF for pH).
[0187] A further technique suitable for determining the amount of CD62L, LFA-
1, CD3, CD4 and/or CD8 on the surface of cells is fluorescence resonance
energy
transfer (FRET). In FRET an excited fluorescent donor molecule, rather than
emitting
light, transfers that energy via a dipole-dipole interaction to an acceptor
molecule in
close proximity. If the acceptor is fluorescent, then the decrease in donor
fluorescence
due to FRET is accompanied by an increase in acceptor fluorescence (i.e., for
example,
sensitized emission). The amount of FRET depends strongly on distance,
typically
decreasing as the sixth power of the distance, so that fluorophores can
directly report on
phenomena occurring on the scale of a few nanometers, well below the
resolution of
optical microscopes. Among other purposes, FRET has been used to map distances
and
study aggregation states, membrane dynamics, or DNA hybridization.
[0188] In principle, FRET measurements can provide information about any
system the components of which can be manipulated to change the proximity of
donors
and acceptors on the scale of a few nanometers. In practice, the ability to
label a system
of interest with appropriate donors and acceptors is constrained by several
physical and
instrumental factors. In addition to the requirement that donor and acceptor
be in close
proximity, the donor emission and acceptor absorption spectra should overlap
significantly with minimal overlap of the direct excitation spectra of the two
fluorophores. Instrumental differences between a fluorescence microscope and a
spectrofluorometer, i.e., spatial confinement of the signal, reduced
sensitivity, and
generally limited wavelength selection, all affect the quality and quantity of
information
that can be extracted from a FRET experiment using a microscope. The use of
FRET in
its traditional incarnation as a molecular ruler to measure absolute distances
is often not
feasible in the fluorescence microscope. Rather, FRET ratio imaging microscopy
is often
used as an indicator of proximity, subject to some degree of calibration.
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[0189] The simplest experimental approach is to excite the donor and measure
both the direct donor emission "DD" and the sensitized emission "DA" of the
acceptor
(the first letter represents the species being excited, and the second letter
represents the
observed emission). The ratio of acceptor-to donor fluorescence, DA/DD, varies
between
two extremes: no energy transfer and maximal energy transfer. When donor and
acceptor
are sufficiently distant, no energy transfer occurs and the donor fluorescence
(DD) is at
its maximum, whereas the sensitized emission is zero. Acceptor fluorescence
results only
from direct excitation of the acceptor, and DA/DD is at its minimum. The
greatest
amount of energy transfer occurs when the donor and acceptor are separated by
the
shortest possible distance, and excited donors lose most of their energy to
the acceptor.
[0190] Complete quantification of FRET can involve significant calculations,
but an estimation of FRET can be obtained easily by measuring the intensity at
two fixed
time points and taking the ratio of these intensities.
[0191] To quantify the relative amount of an acceptor, the acceptor can also
be
excited directly with the wavelength ideal for acceptor fluorescence, so that
"AA" is
recorded rather than DA. With AA used as the reference, the ratio DD/AA can
also be
used as a measure of FRET. Measurement of AA does not generally affect the
measurement of DD because acceptor excitation wavelengths are always longer
(lower
energy) than donor excitation wavelengths, thus avoiding photobleaching of the
donor.
[0192] Although photobleaching should usually be minimized, it can in some
cases actually be exploited to measure FRET. Photobleaching of the donor
usually occurs
when it is in the excited state: before fluorescence emission occurs there is
some
probability that photobleaching will remove that fluorophore from the excited
state, and
also from future excitation emission cycles. When FRET occurs, the donor is
removed
from the excited state before emission or photobleaching, and the bleach rate
decreases
because that donor remains available for another cycle of excitation emission.
The
efficiency of FRET can be determined from the bleach rate of donor
fluorescence in the
presence of acceptor compared with the bleach rate of the donor in the absence
of
acceptor. Experimentally, the instantaneous intensity, 1(t), is normalized to
the initial
intensity 1(0) and the decay of fluorescence intensity is analyzed. A major
advantage of
the photobleaching method is that it uses only a single excitation wavelength
and only a
single emission wavelength. The bleach rate of the donor in the absence of
acceptor
should be measured under experimental conditions identical to those for the
donor-
acceptor pair, because bleaching rates can vary significantly for different
intracellular
environments.
[0193] If (i) the amount of FRET is relatively small; (ii) the acceptor is not
fluorescent; or (iii) rapid photobleaching prevents measurement of static
fluorescence
intensities, a photobleaching method may provide the only practical
measurement of
FRET. In particular, the photobleaching method should be useful with the high
illumination intensities typical with lasers used for confocal microscopy.
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[0194] Determining the level or amount of CD62L, LFA-1, CD4, CD8 and/or
CD3 in the sample typically involves the formation of signals, e.g. signals
generated by a
detectable marker (supra) that can be quantified. Quantifying the signals in
order to
determine the level of e.g. CD62L, CD3 and/or LFA-1 in the sample may be
carried out
by comparing obtained signals with those of one or more reference
measurements. As
will be apparent from the above, the word "comparing" as used herein refers to
a
comparison of parameters or values in terms of absolute amounts/levels that
correspond
to each other. As an example, a number of cells is compared to a reference
number of
cells, a concentration is compared to a reference concentration, or a signal
intensity
obtained from a test sample is compared to the intensity of a corresponding
type of signal
obtained in a reference sample. A respective reference measurement may be
based on the
signal generated by a known amount of CD62L, LFA-1 and/or CD3. Such a known
amount of CD62L, LFA-1 and/or CD3 may for example be present in a sample with
a
composition that resembles the sample from the subject, in which the amount of
CD62L,
LFA-1 and/or CD3 is to be determined. A respective reference sample may be
taken to
define an external reference sample. In some embodiments of a method of the
invention
an internal reference sample may in addition or alternatively be used. Such an
internal
reference sample is a sample obtained from the subject at a previous point of
time. The
amount of CD62L, LFA-1 and/or CD3 in such a sample may be determined to
identify
the changes in CD62L, LFA-1 and/or CD3 levels in the subject. In some
embodiments
the level or amount of CD62L, LFA-1 and CD3, respectively, in the sample may
be
normalized by a comparison to the level of one or more other proteins,
typically cell
surface proteins that are known in the art to be stably expressed. In some
embodiments a
technique of determining the number, amount or ratio of T cells that have e.g.
CD62L
and/or LFA-1 on their surface includes calibrating the analysis equipment. In
embodiments where flow cytometry is used, a standardized blood cell sample may
for
example be used such as the IMMUNO-TROL Control Cells commercially available
from Beckman Coulter Inc. (Fullerton, CA, USA, order No. 6607077).
[0195] In some embodiments of the method of the invention the amount or level
of T cells that have both CD62L and CD3 determined in the sample may be
compared to
a threshold value. In some embodiments of the method of the invention the
amount or
level of T cells that have both LFA-1 and CD3 determined in the sample may be
compared to a threshold value. In some embodiments the amount of T cells that
have
both CD62L and CD3 determined in the sample may be compared to a threshold
value.
In some embodiments the amount of T cells that have CD62L, LFA-1 and CD3
determined in the sample may be compared to a threshold value. In some
embodiments
the amount or level of T cells that have CD62L, LFA-1 and CD3 determined in
the
sample may be compared to a threshold value. In some embodiments the ratio of
T cells
that have CD62L and/or LFA-1 and CD3 to T cells that have only CD3, but not
CD62L
and/or LFA-1, may be determined in the sample may and compared to a threshold
ratio.
In some embodiments the ratio of T cells that have both CD62L and CD3 or both
LFA-1
and CD3 to all T cells that have CD3 may be determined in the sample may and
compared to a threshold ratio. In some embodiments the ratio of T cells that
have
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CD62L, LFA-1 and CD3 determined in the sample may be compared to a threshold
value. In some embodiments the amount or level of T cells that have CD62L as
well as
CD3 determined in the sample may be compared to a threshold value. In some
embodiments the ratio of T cells that have CD62L and/or LFA-1 and CD3 to T
cells that
have only CD3, but not CD62L and/or LFA-1, may be determined in the sample and
may
compared to a threshold ratio. In some embodiments the ratio of T cells that
have
CD62L, LFA-1 and CD3 determined in the sample may be compared to a threshold
value.
[0196] A respective threshold value may in some embodiments be a
predetermined threshold value. In some embodiments the threshold value is
based on the
amount of cells having both CD62L and CD3 in a control sample or both LFA-1
and
CD3 in a control sample. As applicable, in some embodiments the threshold
value is
based on the amount of cells having CD62L, LFA-1 and CD3 in a control sample.
In
some embodiments the threshold value is a threshold ratio based on the ratio
of cells that
have both CD62L and/or LFA-1 and CD3 to T cells that have only CD3, but not
CD62L
and/or not LFA-1, or to all T cells that have CD3 in a control sample. A
respective
control sample may have any condition that varies from the sample main
measurement
itself. Such a control sample may be a sample of, include or essentially
consist of the
corresponding body fluid as the sample from the subject. A control sample may
for
example be a sample, such as a blood sample, a plasma sample, a serum sample
or a
cerebrospinal fluid (liquor) sample, of a subject known not to suffer from
PML. In some
embodiments a respective control sample is from a subject that is age-matched.
In some
embodiments a respective control sample is from a subject that is known not to
have a
confounding disease, in some embodiments from a subject known not to have PML,
or
from a subject known to suffer from MS, as applicable, and in some embodiments
from a
subject known not to have a disease.
[0197] In some embodiments a threshold value is based on a control or
reference
value obtained concomitantly with the value of the sample from the subject. In
some
embodiments a respective control or reference value is determined at a
different point in
time, for example at a point in time earlier than the measurement of the
sample from the
subject is carried out. It is understood that the terms control and reference
may in some
embodiments be a range of values.
[0198] Population studies may also be used to select a threshold value.
Receiver
Operating Characteristic ("ROC") arose from the field of signal detection
theory
developed during World War II for the analysis of radar images, and ROC
analysis is
often used to select a threshold able to best distinguish a diseased
subpopulation from a
nondiseased subpopulation. A false positive in this case occurs when a person
tests
positive, but actually does not have the disease. A false negative, on the
other hand,
occurs when the person tests negative, suggesting the person is healthy, when
it actually
does have the disease. To draw a ROC curve, the true positive rate (TPR) and
false
positive rate (FPR) are determined as the decision threshold is varied
continuously. Since
TPR is equivalent with sensitivity and FPR is equal to 1 - specificity, the
ROC graph is
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sometimes called the sensitivity vs (1 - specificity) plot. A perfect test
will have an area
under the ROC curve of 1.0; a random test will have an area of 0.5. A
threshold is
selected to provide an acceptable level of specificity and sensitivity.
[0199] In addition to threshold comparisons, other methods for correlating
assay
results to a patient classification (occurrence or nonoccurrence of disease,
likelihood of
an outcome, etc.) include decision trees, rule sets, Bayesian methods, and
neural network
methods. These methods can produce probability values representing the degree
to which
a subject belongs to one classification out of a plurality of classifications.
[0200] The comparison to a threshold value, which may be a predetermined
threshold value, can be carried out manually, semi-automatically or in a fully
automated
manner. In some embodiments the comparison may be computer assisted. A
computer
assisted comparison may employ values stored in a database as a reference for
comparing
an obtained value or a determined amount, for example via a computer
implemented
algorithm. Likewise, the comparison to a reference measurement may be carried
out
manually, semi-automatically or in a fully automated manner, including in a
computer
assisted manner.
[0201] The level of expression of CD62L and LFA-1 determined in or from a
sample of a subject may be expressed in terms of cell numbers, i.e. the number
of T cells
that are positive for CD62L and for LFA-1. The level of expression of CD62L
and LFA-
1 may also be expressed in terms of the total amount of CD62L and LFA-1 in a
sample.
As explained above, where immobilization of cells onto a surface is employed,
for
example an immobilized capture probe specific for T cells, the total amount of
CD62L
and LFA-1 present on the respective cells may be used to express the total
amount of
CD62L and LFA-1. In some embodiments a high level of soluble CD62L can be
expected to be included in the sample from a patient. Soluble CD62L originates
for
example from granulocytes. In such embodiments it may be advantageous to
distinguish
soluble CD62L and CD62L present on the surface of cells or to remove soluble
CD62L
before detecting CD62L in the detection method. Whether high levels of soluble
CD62L
are to be expected in a sample can easily be tested by for instance measuring
a single
value of the sample with and/without immobilizing T cells and subsequently
washing the
same. A significant difference of the obtained values indicates a high amount
of soluble
CD62L in the sample. The term "significant" is used to indicate that the level
of decrease
or increase is of statistical relevance, and typically means a deviation of a
value relative
to another value of about 2 fold or more, including 3 fold or more, such as at
least about
to about 10 fold or even more.
[0202] The expression level of CD62L or LFA-1 determined in or from a sample
of a subject can be compared to a single control sample or a plurality of
control samples,
such as a sample from a control subject, in any suitable manner. As an
illustrative
example, the expression level of CD62L or LFA-1 in a control sample can be
characterized by an average (mean) value coupled with a standard deviation
value, for
example at a given time point. In some embodiments the expression level of
CD62L or
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LFA-1 in a subject may be considered different when it is one standard
deviation or more
higher or lower than the average value of the corresponding expression level
determined
in one or more control samples. In some embodiments the determined expression
level of
CD62L or LFA-1 is regarded as different where the obtained value is about 1.5
standard
deviations higher or lower, including about two, about three, about four or
more standard
deviations higher or lower than the average value determined in a control
sample. In
some embodiments the determined expression level of CD62L or LFA-1 is regarded
as
different where the obtained value is about 1.2 times or more higher or lower,
including
about 1.5 times, about two fold, about 2.5-fold, about three fold, about 3.5
fold, about 4-
fold, about 5-fold or more higher or lower than the expression level
determined in a
control sample. In some embodiments the determined expression level of CD62L
or
LFA-1 is regarded as different where the obtained value is about 0.8-fold or
less than the
expression level determined in a control sample. The determined expression
level of
CD62L or LFA-1 may for example be regarded as different if a value is about 70
%, such
as about 60 % or about 50 % lower than the expression level determined in a
control
sample. In some embodiments an expression level of CD62L or LFA-1 is regarded
as
different if the obtained value is about 40 %, including about 30 % lower than
the
expression level determined in a control sample. An expression level of CD62L
or LFA-
1 is in some embodiments regarded as different if the obtained value is about
25 %, such
as about 20 % or lower than the expression level determined in a control
sample.
[0203] In some embodiments a reduced amount of CD62L or LFA-1 relative to a
threshold value, indicates an elevated risk of occurrence of PML in a subject.
An amount
of CD62L or LFA-1 that is not below a threshold value or that is above a
threshold value
indicates that there is no elevated risk of occurrence of PML in the subject.
A level of
CD62L or LFA-1 below a threshold value may indicate a condition where the
subject is
in need of therapy or in need of a change of a therapy to which the subject is
being
exposed. If a level of CD62L or LFA-1 is detected that is above a
predetermined
threshold value, this may indicate that no PML has occurred, as well as that
the risk of
occurrence of PML is low.
[0204] In some embodiments a plurality of measurements is carried out on a
plurality of samples from the same patient. In each of the samples the level
of expression
of CD62L or LFA-1 is determined. Typically the level of expression determined
in each
of the samples is compared to a threshold value as detailed above. In some
embodiments
the plurality of samples from the same individual is taken over a period of
time at certain
time intervals, including at predetermined time intervals. Such an embodiment
may be
taken as a method of monitoring the expression of CD62L and optionally LFA-1.
Matching samples may in some embodiments be used to determine a threshold
value for
each corresponding time point. The average value may be determined and the
standard
deviation calculated for each given time point. A value determined in the
sample from
the subject falling outside of the mean plus 1 standard deviation may be
indicative of
susceptibility to PML.
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[0205] In one embodiment the level of CD62L or LFA-1 is measured at certain,
e.g. predetermined, time intervals. Samples from the subject may be provided
that have
been obtained at the corresponding time points. As an illustrative example,
samples may
be taken from the same subject after a time interval of about 3 months,
including about
every month. In some embodiments samples may be taken from the same subject at
a
time interval of about 6 months. In some embodiments a sample may be taken
from the
same subject after a time interval of about a year, i.e. about 12 months. In
some
embodiments a sample may be taken from the same subject after about 18 months.
A
value obtained from a respective sample may in some embodiments be compared to
a
sample taken from the same subject at a previous point of time, for example
the previous
measurement and/or the first measurement taken. In this way a change in the
level of
CD62L or LFA-1 may be detected. Matching samples may in some embodiments be
used to determine a threshold value for each corresponding time point. The
average value
may be determined and the standard deviation calculated for each given time
point. As an
illustrative example, a value determined in the sample from the subject
falling outside of
the mean plus 1 standard deviation may for instance be indicative of the
occurrence or of
the risk of occurrence of PML.
[0206] In some embodiments a measurement is repeated if during monitoring,
i.e. measuring the amount of CD62L or LFA-1 at certain time intervals an
increase or
decrease is detected, in particular if an increase or decrease beyond a
threshold value is
detected. In some embodiments time intervals after which the level of CD62L or
LFA-1
are being determined may be shortened if during monitoring of the amount of
CD62L or
LFA-1 an increase or decrease has been detected. As an illustrative example, a
decrease
or increase in levels of one or more of CD62L or LFA-1 may have been found at
a
certain point of time during measurements carried out at intervals of 12
months or during
measurements carried out at intervals of 18 months. After such a decrease or
increase in
levels has been found, monitoring of the level of CD62L or LFA-1 may be
continued at
time intervals of about a month. As indicated above, monitoring the amount of
CD62L or
LFA-1 may be included in the context of monitoring a therapy, for example in
order to
assess the efficacy thereof or to evaluate a subject's response to a certain
treatment.
[0207] The terms "treatment" and "treating" as used herein, refer to having a
therapeutic effect and at least partially alleviating or abrogating an
abnormal condition in
the organism of a subject. Generally a treatment reduces, stabilizes, or
inhibits
progression of a symptom that is associated with the presence and/or
progression of a
disease or pathological condition. The term "administering" relates to a
method of
incorporating a compound into cells or tissues of a subject. The term
"therapeutic effect"
refers to the inhibition or activation of factors causing or contributing to
the abnormal
condition. A therapeutic effect relieves to some extent one or more of the
symptoms of
an abnormal condition or disease. The term "abnormal condition" refers to a
function in
the cells or tissues of an organism that deviates from their normal functions
in that
organism. An abnormal condition can inter alia relate to cell proliferation,
cell
differentiation, or cell survival.
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[0208] In embodiments where the subject is to be treated, for example with a
VLA-blocking agent, monitoring expression levels may in some embodiments start
prior
to the treatment. In some embodiments monitoring may start at the same time or
at an
early stage of the treatment, e.g. administration of VLA-4 blocking agents.
[0209] As indicated above, in some embodiments a method according to the
present invention includes measuring CD62L and optionally LFA-1 expression on
T cell
in a sample or obtained from a sample, and comparing the result obtained
therefrom to a
reference value. In the context of a therapy in some embodiments detecting the
level of
CD62L expressing T cells as well as monitoring the same includes determining
whether
one or more of the following indications is present:
[0210] (1) In the context of therapy with a VLA-4 blocking agent, a lack of
CD62L expression may be observed after administration of a VLA-4 blocking
agent. The
lack of CD62L expression may be observed at a point of time, such as within
about the
first week. In some embodiments lack of CD62L expression may be detected
within
about the second week or within about the third week. A lack of CD62L
expression may
in some embodiments be detected within about the 1st month, within about 2hd
month,
th, 5th, 6th, 7th, 8th, 9th, 10th, 1 th, 12th, 13th, 14th,
within about 3rd month, within about the 4
15th or within about the 16th month. In some embodiments a lack of CD62L
expression
may be detected in the 17th month. In some embodiments lack of CD62L
expression may
be detected in the 18th month. A lack of CD62L expression may in some
embodiments be
detected within the 19th, the 20th, 21st, 22hd, 23rd, 24th, - -th
2.J month or longer. For example,
Fig. 8 shows that only very low levels of CD62L in a sample from one subject
who later
developed PML could be detected after 15 months of treatment.
[0211] (2) A differential expression level of CD62L on the T cell surface
compared to a reference level obtained from "control subjects", as indicated
above. A
differential expression in some embodiments refers to a "decreased" expression
compared to a reference. In the context of therapy with a VLA-4 blocking agent
control
subjects may be defined as those who underwent VLA-4 blocking agent treatment
for
about one year or more, such as about 1.5 years or more, about 2 years or
more, or about
3 years or longer but who have not been diagnosed with PML. The samples to be
compared are in some embodiments obtained from the same or substantially the
same
time point after the initiation of the treatment. For example, a 1-month
sample is in some
embodiments compared to another 1-month sample. In the context of HIV
infection a
control subject may be an individual of a comparable stage of AIDS, who is
known not
to have PML. A "differential" expression is observed by comparing a measured
expression level to a corresponding level of one or more control subjects. A
"differential" expression, as used throughout the present application, is
observed when
the expression is lower or higher than that observed from one or more control
subjects
such that one of skill in the art would consider it to be of statistical
significance. In such
case, the differential expression is a "decreased" expression compared to a
reference.
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[0212] The expression of CD62L determined from a sample of a subject can be
compared to control patients in any suitable manner. For example, the
expression of
CD62L in the control patient can be characterized by an average (mean) value
coupled
with a standard deviation value at a given time point. The expression of CD62L
in a
subject may be considered different when it is more than one standard
deviation different
from the average value.
[0213] As used herein, the term "level" is intended to mean the amount,
accumulation or rate of synthesis of the biomarker molecule. A level can be
represented,
for example, by the amount or synthesis rate of messenger RNA (mRNA) encoded
by a
gene, the amount or synthesis rate of polypeptide corresponding to a given
amino acid
sequence encoded by a gene, or the amount or synthesis rate of a biochemical
form of a
molecule accumulated in a cell, including, for example, the amount of
particular post-
synthetic modifications of the nucleic acid or polypeptide. The term can be
used to refer
to an absolute amount of the biomarker protein or mRNA in a sample or to a
relative
amount of the molecule, including amounts determined under steady-state or non-
steady-
state conditions. Thus, a CD62L expression level may be represented by the
amount of
CD62L, accumulation or rate of synthesis of CD62L or the precursor form or a
post-
translationally modified form of CD62L.
[0214] (3) A low number of T cells expressing CD62L. This can be represented
by, for example, ratio of such cells to total PBMC, number of cells per sample
(e.g. mm3
blood), ratio of such cells to all T cells, or otherwise, as suitable for such
representation.
When the number is represented by percentage of T cells expression CD62L, a
"low"
percentage is defined as less than about 10%. In some embodiments a low
percentage is
defined as less than about 9%, such as less than about 8%, such as less than
about 7%,
6%, 5%, 4%, or 3%. A low percentage of T cells expressing CD62L is in some
embodiments defined as less than about 2%. %. In some embodiments a low
percentage
is defined as 1% or less, including about 0.5%, or less. If other methods are
employed, a
skilled person is able to convert the values here given according to the
method used and
common knowledge. If the value observed is persistently low over an extended
period of
time, the subject is more susceptible to PML. "Extended period of time" refers
to a
period of a plurality of months, such as about 5 months or more. In some
embodiments
an extended period of time is a period of 6 months or more, such as about 7,
8, 9, 10, or
11 months. In some embodiments an extended period of time is a period of 12
months or
longer. As an illustrative example, Fig. 8 shows that the CD62L levels on T
cells from
subjects who later developed PML were persistently low.
[0215] (4) A lack of "recovery" of the percentage of T cells which expresses
CD62L. The term "recovery" is determined by comparing the obtained amount or
level
to a threshold value, which may be based on a reference level. As used herein,
"recovery" is defined as a return of the percentage of T cells which express
CD62L back
to the range of the reference level or higher.
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[0216] The reference level for this purpose can be determined by various
methods. In some embodiments, the reference is obtained from the same subject
at the
first month of the treatment of VLA-4 blocking agent. In some embodiments, the
reference level may be from an earlier point in time, such as about 3 months
ago. In some
embodiments the earlier point in time may be 4 months ago, such as about 5
months ago.
The earlier point in time may in some embodiments be about 6 months ago. In
some
embodiments the earlier point in time may be about 7 or about 8 months ago, or
historical reference level from past course of treatment. In some embodiments,
the
reference level is obtained from one or more control subjects, such as about
30 or more
control subjects who underwent treatment of VLA-4 blocking agent for more than
1 year.
The reference level is in some embodiments obtained from about 40 or more
control
subjects, including about 50 or more, about 60 or more or about 70 or more
control
subjects who underwent treatment of VLA-4 blocking agent for more than 1 year,
such
as more than about 1.5 years, more than about 2 years, about 2.5 years, about
3 years, or
more. In some embodiments, the reference level is measured within about the
first month
after the first administration of the VLA-4 blocking agent.
[0217] Fig. 8 shows that the CD62L levels of control subjects recovered
(exceeding the reference level taken at the first month) after 15 months of
VLA-4
blocking agent treatment.
[0218] In some embodiments, a sample from the subject to be tested is taken
about one month after the treatment. PMBC is isolated from the sample and
subjected to
a suitable detection technique such as FACS analysis. The percentage of T
cells,
including CD4 T cells and/or CD8' T cells, which are CD62L positive is
measured and
compared to a reference level derived from one or more control subjects. If
the measured
value is lower or higher than the threshold value, it is indicative of an
increased
susceptibility to PML.
[0219] For instance, the following indicate reference levels for CD62L that
can
be used to set a threshold value:
Month % of CD4' T cells reference level (mean % of
positive for CD62L (mean CD4 'CD62L ' T cells minus
(standard deviation)) 1 standard deviation)
0 (before treatment) 53.7 (10.2) 43.5
1 28.2 (7.2) 21.0
3 36.9 (17.8) 19.1
6 20.0 (14.9) 5.1
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12 16.7 (16.2) 0.5
15-20 40.0 (13.9) 26.1
21-25 41.8 (10.3) 31.5
26-30 44.9 (12.5) 32.4
31-35 34.0 (6.9) 27.1
36-40 38.0 (18.1) 19.9
41-45 33.5 (6.4) 27.1
46-50 32.3 (2.3) 30.0
51-55 39.5 (20.7) 18.8
Table 1: Exemplary reference values for CD62L for individuals receiving
Natalizumab
[0220] By way of example, the reference level for a subject having received 1
month treatment of Natalizumab may be 21%. An expression level lower than 21%
may
be considered "different" and indicate a risk for PML.
[0221] When one of the above indications is observed, for example, when there
is a lack of CD62L expression, or when low expression of CD62L persists for an
extended period of time, the physician should consider, combined with other
information
available, stopping or temporarily withholding the treatment, adjusting the
dosage,
performing plasma exchange, or the like until the expression level increases
or recovers.
It may be possible to resume the treatment after the expression level of the
biomarkers in
the present invention has recovered or increased.
[0222] As can be taken from Fig. 3 and Fig. 8, levels of CD62L on T cells
tend,
with the exception of about the initial 12 months of treatment at all, to
remain within a
relatively stable range during treatment of relapsing remitting multiple
sclerosis with a
VLA-4 blocking agent. A drop of CD62L on T cells can typically be observed
after onset
of PML (cf. also Fig. 8). It is thus in some embodiments helpful to monitor
the time
course of CD62L levels on T cells of an individual. In this way any unexpected
alteration
of CD62L levels can be detected. Such alteration is an indication of an
elevated risk of
PML.
[0223] As explained above, in some embodiments of a method or use of the
invention the expression level of LFA-1 in the sample is determined. In some
embodiments the expression level of LFA-1 is determined at a plurality of time
points,
for example by determining the expression level of LFA-1 in a plurality of
samples,
which have been obtained from the same subject at particular time points over
a period of
time. If the expression level of LFA-1 observed is persistently different from
a threshold
value over an extended period of time, the subject is more susceptible to PML.
As an
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example in this regard, Fig. 7 shows that that the LFA-1 levels of two
patients who later
developed PML were persistently lower than that from control patients after
month 6 and
12.
[0224] For instance, a reference level of LFA-1 as indicated in the following
can
be used to set a threshold value:
Month % of CD4-' T cells positive for reference level (mean % of
LFA-1 (mean (standard CD4+LFA-1 T cells minus 1
deviation)) standard deviation)
0 (before 35.5(13.6) 21.9
treatment)
1 31.2 (12.7) 18.5
3 25.4 (7.6) 17.8
6 23.8 (9.8) 14.0
12 28.9 (10.2) 18.7
15-20 47.3 (16.3) 31.0
21-25 59.9 (7.5) 52.4
26-30 50.4 (16.8) 33.6
31-35 26.0 (19.8) 6.2
36-40 40.6 (18.9) 21.7
41-45 37.0 (5.7) 31.3
46-50 34.5 (11.8) 22.7
51-55 42.2 (17.2) 25.0
Table 2: Exemplary reference values for LFA-1 for individuals receiving
Natalizumab
[0225] For the purpose of the present invention, the detection of LFA-1
expression can also include detecting the protein or mRNA of CD1 1 a and
Runx3. In this
case, the determination of susceptibility may be carried out using generally
the same
approach for LFA-1 protein.
[0226] As should be apparent from the above, if for example a level of T cells
that have CD62L and/or both CD62L and LFA-1 is detected that is above or below
a
(e.g. predetermined) threshold value, this may indicate a risk that the
subject will have
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PML, generally at a later point of time. In embodiments where the sample is
from an
HIV positive subject, if a level of T cells that have both CD62L and LFA-1 is
detected
that is above or below a predetermined threshold value, this may indicate the
need to
change therapy. A level of T cells that have both CD62L and LFA-1 above or
below a
predetermined threshold value may also indicate a condition where the subject
is
suffering from PML. In case it is suspected that a subject is suffering from
PML the
practitioner will usually carry out MRI imaging. It may for example be
analysed whether
lesions in subcortical white matter exist. The presenting PML symptoms most
commonly
include changes in cognition, behaviour, and personality, but in some cases
seizures may
be the first clinical event. Such symptoms may occur either alone or
associated with
motor, language, or visual symptoms.
[0227] If the expression level of CD62L on T cells from a subject is detected
that is above or below a threshold value, this may also indicate a risk that
the subject will
have PML. If the expression level of CD62L observed is for instance
persistently
different from a threshold value over an extended period of time, the subject
is diagnosed
to be at an elevated risk to develop PML.
[0228] In some embodiments of a method according to the invention prior to a
planned treatment the level of CD62L on T cells from a subject is determined
as detailed
above. If an increased or a decreased level of CD62L present on T cells,
relative to a
threshold value, is determined, an increased risk of PML occurrence may be
diagnosed.
In embodiments where the subject is HIV positive the planned therapy may be
adjusted
in order to achieve a particularly fast and effective immune restoration and/
or in order to
assist the subject's organism to provide JCV specific T cell responses. In
some
embodiments it may be considered to include a HT2a antagonist into a planned
therapy.
As indicated above, in some embodiments the level of CD62L on T cells from a
subject
may be monitored over time. For this purpose frozen samples that were obtained
from
the subject at different time points may for instance be analysed within the
same
measurement. The level of CD62L on T cells may for instance be measured at
time
intervals of one or more months such as about every 6 months, about every 8
months,
about every 10 months, about every 12 months or about every 14 months during a
treatment, for instance with a VLA-4 blocking agent, or as long as the subject
is
diagnosed to suffer from a disease such as HIV or multiple sclerosis. A change
of the
level of CD62L on T cells may indicate that the subject is at a risk of
developing PML.
Depending on further diagnosis results, a change of the level of CD62L on T
cells may
also indicate that the subject is developing PML. In some embodiments where
the subject
is undergoing treatment with a VLA-4 blocking agent the level of CD62L on T
cells may
be determined before a treatment with a VLA-4 blocking agent is begun.
Thereafter a
further analysis of the level of CD62L on T cells may for instance be carried
out about
1.5 years after the start of treatment. Subsequently the level of CD62L on T
cells from
the subject may be analysed about every 6 months.
[0229] Methods according to the present invention can be used to predict
whether a subject is likely to develop PML. This is of particular importance
since no
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PML therapy is currently available and overall mortality is above 50 %, as
explained
above. In addition, once PML is diagnosed in a subject undergoing treatment
with a
VLA-4 blocking agent, plasma exchange or immunoadsorption is required in order
to
more rapidly remove the VLA-4 blocking agent from plasma and to speed up the
reconstitution of immune surveillance. In this regard immunoadsorption is only
established as a medical procedure in Europe and Japan, but not in North
America. The
reconstitution of immune function following removal of e.g. a monoclonal
immunoglobulin with plasma exchange procedures, or immune reconstitution with
HAART, is often accompanied by an exaggerated pathological inflammatory
response
termed immune reconstitution inflammatory syndrome (IRIS), also known as
"restoration disease (IRD)", "immune reconstitution syndrome (IRS)", "immune
recovery disease", and "immune rebound illness". As the immune system
recovers,
influx of cytotoxic and bystander lymphocytes eliminates infected
oligodendrocytes and
augments bystander inflammation. The immune system has been postulated to
respond to
a previously acquired opportunistic infection with an overwhelming
inflammatory
response that paradoxically renders symptoms of infection worse. Since IRIS
has been
found to occur in the absence of any apparent active infection, it has also
been postulated
to arise merely due to restoration of the previously suppressed inflammatory
immune
response due to reactivation of memory cells that had been previously
activated by
antigen exposure. IRIS typically leads to clinical deterioration, causing high
disability
and mortality. IRIS was first described in patients with HIV, however it is
more common
in MS patients treated with Natalizumab.
[0230] In HIV infected subjects IRIS typically develops within weeks or months
(Post, M.J.D., et al., Am. J. Neuroradiol. (2013) 10.3174/ ajnr.A3183). IRIS
significantly
negatively impacts the HIV infected population on HAART by increasing the
number of
procedures, number of hospitalizations, and the overall morbidity in this
patient cohort
(ibid.). Among JCV positive HIV infected patients that have been treated with
HAART,
it has been reported that 18 % may develop IRIS (ibid.). In HIV negative
patients on
immunomodulatory therapy such as natalizumab, PML-IRIS is reportedly more
severe
than in HIV infected patients due to the restored immune surveillance in the
latter (ibid.).
[0231] IRIS is a robust inflammatory response, which may occur as a mild
disease, but also as a life-threatening deterioration. A method according to
the invention
allows early prediction of the risk of PML occurrence and therefore provides
time to
adjust treatment before onset of PML. Thus occurrence of IRIS may be avoided
and
thereby a potential additional health/life risk be circumvented.
[0232] A method as described above may also be a method of assessing the
occurrence of PML. In such an embodiment the subject from whom/which the
sample
originates is generally suspected to suffer from PML. An increased amount of
CD62L
and/or LFA-1, relative to the threshold value, indicates the presence of PML.
A method
as described above may further in some embodiments be a method of assessing
the
chances of survival from PML in a subject. The subject is generally known to
have PML.
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An increased or decreased level of CD62L expressing T cells, relative to the
threshold
value, may indicate low chances of survival of PML.
[0233] The present invention further provides a method of treating a subject
having a demyelinating disease or an autoimmune disease. The method includes
administering a VLA-4 blocking agent to the subject. Typically the method also
includes
monitoring the expression of at least one biomarker on T cells, with the
monitoring being
carried out on a sample from the subject. A respective biomarker may be CD62L
and/or
LFA-1. The method may also include determining the migratory capacity of
CD45 'CD49d immune cells. The VLA-4 blocking agent can be used to treat a
number
of diseases and disorders, including multiple sclerosis, Crohn's disease,
rheumatoid
arthritis, meningitis, neuromyelitis optica, neurosarcoidosis, CNS vasculitis,
chronic
inflammatory demyelinating polyradiculo-neuropathy (CIDP), encephalitis,
transverse
myelitis, tissue or organ graft rejection or graft-versus-host disease,
chronic renal
disease, CNS injury, e.g., stroke or spinal cord injury; chronic renal
disease; allergy, e.g.,
allergic asthma, type 1 diabetes, an inflammatory bowel disorder, e.g.,
ulcerative colitis,
myasthenia gravis, fibromyalgia, arthritic disorders, e.g., rheumatoid
arthritis or psoriatic
arthritis, an inflammatory/immune skin disorder, e.g., psoriasis, vitiligo,
dermatitis or
lichen planus, systemic lupus erythematosus, Sjogren's Syndrome, a
hematological
cancer, e.g., multiple myeloma, leukemia or lymphoma, a solid cancer, e.g., a
sarcoma or
a carcinoma, e.g., of the lung, breast, prostate, brain, as well as a fibrotic
disorder, e.g.,
pulmonary fibrosis, myelofibrosis, liver cirrhosis, mesangial proliferative
glomerulonephritis, crescentic glomerulonephritis, diabetic nephropathy, and
renal
interstitial fibrosis. Any disease or pathological condition which has been
treated or is
known to be treatable by the blocking agent may be treated in the context of
the present
invention.
[0234] Typically the treatment of a subject having a demyelinating disease or
an
autoimmune disease includes administering a therapeutically effective amount
of a VLA-
4 blocking agent. As used herein, the term therapeutically effective amount of
VLA-4
blocking agent is an amount sufficient to provide a therapeutic benefit in the
treatment or
management of the relevant pathological condition, or to delay or minimize one
or more
symptoms associated with the presence of the condition. The blocking agent
may, for
example, be administered intravenously. For Natalizumab, the dose may be 1 to
6 mg per
kilogram of body weight. In one embodiment, a standard does of 300 mg
Natalizumab
diluted with 100 ml 0.9% sodium chloride is injected intravenously once every
four
weeks. The dose may be repeated at intervals from two to eight weeks. For
example, a
treatment regimen may include 3 mg Natalizumab per kg body weight repeated at
about
a four week interval. A skilled person in the art is capable of determining
the therapeutic
effective amount and the methods are also described in the references
incorporated
herein.
[0235] A subject may be first subjected to prior screening to determine
whether
the planned treatment would be suitable. For example, such a screening may be
based on
the patient history, previous use of immunosuppressant, Expanded Disability
Status
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Scale (EDSS) in case of multiple sclerosis patients, anti-JCV antibody status
(JCV
antibody seropositivity), MRI imaging studies, pre-infusion checklist for
continuously
worsening neurological symptoms, and other criteria commonly used.
[0236] Typically a subject undergoing VLA-4 blocking agent treatment is tested
to determine the expression level of a biomarker as disclosed herein, e.g. the
expression
level of CD62L on T cells in or from a sample of the subject. As a further
example, the
migratory capacity of CD45 'CD49d immune cells may be determined. A method
according to the invention may also include any other molecule or effect that
can be used
to indirectly indicate the level of such biomarker. One or more samples from
the subject
are collected and analyzed. In some embodiments the one or more samples are
sent to a
central testing facility to ensure that the analysis of phenotype and function
can be
carried out under standardized conditions. Samples may be taken and tested
prior to the
treatment and then regularly after the treatment begins, such as monthly,
bimonthly,
quarterly, every six months, and yearly. The routine assessment for PML
provides timely
information regarding the safety issues related to the treatment. In one
embodiment, the
samples are taken at month 1, every 3 months until the first year, and then
every 6
months thereafter.
[0237] In some embodiments treating the subject undergoing treatment with a
VLA-4 blocking agent includes determining, including monitoring, the
expression level
of CD62L and/or LFA-1 on T cells in or from a sample of the subject. If any
indication is
found that suggests an increased susceptibility to PML or other complications,
or renders
such complication more likely than in other subjects, further tests may be
carried out.
Subjects showing compromised immune surveillance should be clinically
monitored very
closely. The physician may test the patient for further biomarkers such as
those provided
in the present invention or known biomarkers, such as anti-JCV antibody status
or other
clinical or MRI criteria. Based on the information, the practitioner will
assess whether to
continue, restart or stop the treatment of VLA-4 blocking agents. The
information
provides significant information to the physician regarding the risk
associated with the
treatment, so that informed benefit-risk decisions can be made accordingly. As
an
illustrative example, the monitoring may at the beginning include only
determining the
level of CD62L expression. When the result indicates an alteration, including
a low
expression level, compared to the reference value as described above, the LFA-
1
expression level and/or the migration capacity of T cells may be tested.
[0238] In some embodiments, a reference value or level can also be gathered
from patients who suffered from PML as a result of VLA-4 blocking agent
treatment.
Expression levels of the biomarkers from the PML patients are recorded over a
period of
time, such as over 2-3 years. Average expression levels, standard deviation,
and relative
standard deviation at given times are calculated for the patients to determine
a range of
expression levels associated with PML patients. When test result from a
patient to be
evaluated is collected, it will be compared to the reference value.
Statistical differences
between the test result and the reference will be determined to identify
significant
variances in between.
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[0239] Accordingly, determining the expression level of CD62L and/or LFA-1
can be used to stratify a subject undergoing or about to undergo treatment
with a VLA-4
blocking agent for suspension of the treatment. The terms "stratifying" and
"stratification" as used herein indicate in one aspect that individuals are
assigned to
groups with similar characteristics such as at a similar risk level of
developing PML. As
an illustrative example, individuals may be stratified into risk categories.
The terms
"stratifying" and "stratification" as used herein indicate in another aspect
that an
individual is assigned to a certain group according to characteristics
matching the
respective group such as a corresponding risk level of developing PML. The
groups may
be, for example, for testing, prescribing, suspending or abandoning any one or
more of a
drug, surgery, diet, exercise, or intervention. Accordingly, in some
embodiments of a
method or use according to the invention a subject may be stratified into a
subgroup of a
clinical trial of a therapy. As explained in the forgoing, in the context of
the present
invention CD62L and/or LFA-1 may be used for PML risk stratification.
[0240] The terms "stratifying" and "stratification" according to the invention
generally include identifying subjects that require an alteration of their
current or future
therapy. The term includes assessing, e.g. determining, which therapy a
subject likely to
suffer from PML is in need of. Hence, in the context of the present invention
stratification may be based on the probability (or risk) of developing PML. A
method or
use according to the invention may also serve in stratifying the probability
of the risk of
PML or the risk of any PML related condition for a subject. A method of
stratifying a
subject for PML therapy according to the invention includes detecting the
amount of
determining the expression level of CD62L and/or LFA-1 as described above,
and/or
assessing the migratory capacity of CD45 'CD49d immune cells of the subject.
As
explained above, in some embodiments on a general basis a CD62L and/or a LFA-1
capture probe can be advantageously used to screen risk patients which have
higher
susceptibility to PML.
[0241] In this regard the use of biomarkers for stratification of patients is
a well-
established procedure in the art. This procedure includes or consists of
linking one or
more patient subpopulations, characterized by a certain feature, in the
context of the
present invention the expression level of a particular protein or migratory
capacity of
cells, to a particular treatment. The general aim of stratification is to
match patients with
therapies that are more likely to be effective and safe. In a more general
context
stratifying patients may include evaluation of patient history and physical
assessment,
combined with laboratory tests on the basis of a method of the present
invention, and
clinical observation. It is understood that stratifying patients is only
feasible if multiple
treatment options with heterogeneous responses for the disease exist. In the
context of
the present invention HIV therapy may be adjusted or treatment with a VLA-4
blocking
agent be suspended for a certain period of time, such as one or more months. A
general
overview of patient stratification and stratified medicine has been given by
Trusheim,
M.R., et al., Nature Reviews Drug Discovery (2007) 6, 4, 287-293.
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[0242] The present invention also provides a method of treating a subject. The
method includes administering a VLA-4 blocking agent to the subject. The
method
further includes determining the expression of one or more biomarkers on one
or more T
cells from the subject, such as CD4 ' T cells or CD8 T cells. The biomarker is
generally
CD62L or LFA-1. In some embodiments the expression of one or more biomarkers
on
one or more T cells from the subject may be monitored. In some embodiments the
method further includes determining, including monitoring, the migration of
CD45 'CD49d immune cells.
[0243] The term "administering", as used herein, refers to any mode of
transferring, delivering, introducing, or transporting matter such as a
compound, e.g. a
pharmaceutical compound, or other agent such as an antigen, to a subject.
Modes of
administration include oral administration, topical contact, intravenous,
intraperitoneal,
intramuscular, intranasal, or subcutaneous administration (cf. below).
[0244] A VLA-4 blocking agent or an antiviral agent can be administered to a
human patient per se, or in pharmaceutical compositions where they are mixed
with other
active ingredients, as in combination therapy, or suitable carriers or
excipient(s).
Exemplary routes include, but are not limited to, oral, transdermal, and
parenteral
delivery.
[0245] Suitable routes of administration may, for example, include depot,
oral,
rectal, transmucosal, or intestinal administration; parenteral delivery,
including
intramuscular, subcutaneous, intravenous, intramedullary injections, as well
as
intrathecal, direct intraventricular, intraperitoneal, intranasal, or
intraocular injections.
[0246] Alternately, one may administer the compound in a local rather than
systemic manner, for example, via injection of the compound directly into a
solid tumor,
often in a depot or sustained release formulation. Furthermore, one may
administer the
drug in a targeted drug delivery system, for example, in a liposome coated
with a blood-
cell specific antibody. The liposomes will be targeted to and taken up
selectively by the
respective cells.
[0247] Pharmaceutical compositions that include the compounds of the present
invention may be manufactured in a manner that is itself known, e.g., by means
of
conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying,
encapsulating, entrapping or lyophilizing processes.
[0248] Pharmaceutical compositions for use in accordance with the present
invention thus may be formulated in conventional manner using one or more
physiologically acceptable carriers including excipients and auxiliaries that
facilitate
processing of the active compounds into preparations that can be used
pharmaceutically.
Proper formulation is dependent upon the route of administration chosen.
[0249] For injection, the agents of the invention may be formulated in aqueous
solutions, for instance in physiologically compatible buffers such as Hanks's
solution,
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Ringer's solution, or physiological saline buffer. For transmucosal
administration,
penetrants appropriate to the barrier to be permeated are used in the
formulation. Such
penetrants are generally known in the art.
[0250] For oral administration, the compounds can be formulated readily by
combining the active compounds with pharmaceutically acceptable carriers well
known
in the art. Such carriers enable the compounds of the invention to be
formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like,
for oral ingestion by a patient to be treated.
[0251] Pharmaceutical preparations for oral use can be obtained by adding a
solid excipient, optionally grinding a resulting mixture, and processing the
mixture of
granules, after adding suitable auxiliaries, if desired, to obtain tablets or
dragee cores.
Suitable excipients are, in particular, fillers such as sugars, including
lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as, for example, maize
starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or
polyvinylpyrrolidone (PVP).
[0252] If desired, disintegrating agents may be added, such as the cross-
linked
polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0253] Dragee cores are provided with suitable coatings. For this purpose,
concentrated sugar solutions may be used, which may optionally contain gum
arabic,
talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide,
lacquer solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or
pigments may be added to the tablets or dragee coatings for identification or
to
characterize different combinations of active compound doses.
[0254] Pharmaceutical preparations that can be used orally include push-fit
capsules made of gelatin, as well as soft, sealed capsules made of gelatin and
a
plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain
the active
ingredients in admixture with filler such as lactose, binders such as
starches, and/or
lubricants such as talc or magnesium stearate and, optionally, stabilizers. In
soft capsules,
the active compounds may be dissolved or suspended in suitable liquids, such
as fatty
oils, liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
All formulations for oral administration should be in dosages suitable for
such
administration. For buccal administration, the compositions may take the form
of tablets
or lozenges formulated in conventional manner.
[0255] For administration by inhalation, the compounds for use according to
the
present invention are conveniently delivered in the form of an aerosol spray
presentation
from pressurized packs or a nebuliser, with the use of a suitable propellant,
e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon
dioxide or other suitable gas. In the case of a pressurized aerosol the dosage
unit may be
determined by providing a valve to deliver a metered amount. Capsules and
cartridges of
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e.g. gelatin for use in an inhaler or insufflator may be formulated containing
a powder
mix of the compound and a suitable powder base such as lactose or starch.
[0256] The compounds may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion. Formulations for
injection may
be presented in unit dosage form, e.g., in ampules or in multi-dose
containers, with an
added preservative. The compositions may take such forms as suspensions,
solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory agents such
as
suspending, stabilizing and/or dispersing agents.
[0257] Pharmaceutical formulations for parenteral administration include
aqueous solutions of the active compounds in water-soluble form. Additionally,
suspensions of the active compounds may be prepared as appropriate oily
injection
suspensions. Suitable lipophilic solvents or vehicles include fatty oils such
as sesame oil,
or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or
liposomes. Aqueous
injection suspensions may contain substances that increase the viscosity of
the
suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the
suspension may also contain suitable stabilizers or agents that increase the
solubility of
the compounds to allow for the preparation of highly concentrated solutions.
[0258] Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before
use. The
compounds may also be formulated in rectal compositions such as suppositories
or
retention enemas, e.g., containing conventional suppository bases such as
cocoa butter or
other glycerides.
[0259] In addition to the formulations described previously, the compounds may
also be formulated as a depot preparation. Such long acting formulations may
be
administered by implantation (for example subcutaneously or intramuscularly)
or by
intramuscular injection. Thus, for example, the compounds may be formulated
with
suitable polymeric or hydrophobic materials (for example as emulsion in an
acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives, for example,
as a
sparingly soluble salt.
[0260] A pharmaceutical carrier for the hydrophobic compounds of the invention
is a co-solvent system including benzyl alcohol, a non-polar surfactant, a
water-miscible
organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-
solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the non-
polar
surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to
volume in
absolute ethanol. The VPD co-solvent system (VPD: D5W) consists of VPD diluted
1:1
with a 5% dextrose in water solution.
[0261] This co-solvent system dissolves hydrophobic compounds well, and itself
produces low toxicity upon systemic administration. Naturally, the proportions
of a co-
solvent system may be varied considerably without destroying its solubility
and toxicity
characteristics.
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[0262] Furthermore, the identity of the co-solvent components may be varied:
for example, other low-toxicity non-polar surfactants may be used instead of
polysorbate
80; the fraction size of polyethylene glycol may be varied; other
biocompatible polymers
may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars
or
polysaccharides may substitute for dextrose.
[0263] Alternatively, other delivery systems for hydrophobic pharmaceutical
compounds may be employed. Liposomes and emulsions are well known examples of
delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents
such as
dimethylsulfoxide also may be employed, although usually at the cost of
greater toxicity.
Additionally, the compounds may be delivered using a sustained-release system,
such as
semipermeable matrices of solid hydrophobic polymers containing the
therapeutic agent.
Various types of sustained-release materials have been established and are
well known
by those skilled in the art. Sustained-release capsules may, depending on
their chemical
nature, release the compounds for a few weeks up to over 100 days. Depending
on the
chemical nature and the biological stability of the therapeutic reagent,
additional
strategies for protein stabilization may be employed.
[0264] A pharmaceutical composition also may include suitable solid or gel
phase carriers or excipients. Examples of such carriers or excipients include
but are not
limited to calcium carbonate, calcium phosphate, various sugars, starches,
cellulose
derivatives, gelatin, and polymers such as polyethylene glycols.
[0265] Pharmaceutical compositions suitable for use in the present invention
include compositions where the active ingredients are contained in an amount
effective
to achieve its intended purpose. More specifically, a therapeutically
effective amount
means an amount of compound effective to prevent, alleviate or ameliorate
symptoms of
disease or prolong the survival of the subject being treated. Determination of
a
therapeutically effective amount is well within the capability of those
skilled in the art,
especially in light of the detailed disclosure provided herein.
[0266] For any compound used in the methods of the invention, the
therapeutically effective dose can be estimated initially from cell culture
assays. For
example, a dose can be formulated in animal models to achieve a circulating
concentration range that includes the IC50 as determined in cell culture
(i.e., the
concentration of the test compound which achieves a half-maximal inhibition of
the
desired activity). Such information can be used to more accurately determine
useful
doses in humans.
[0267] Toxicity and therapeutic efficacy of the compounds described herein can
be determined by standard pharmaceutical procedures in cell cultures or
experimental
animals, e.g., for determining the LD50 (the dose lethal to 50% of the
population) and
the ED50 (the dose therapeutically effective in 50% of the population). The
dose ratio
between toxic and therapeutic effects is the therapeutic index and it can be
expressed as
the ratio between LD50 and ED50. It may be desired to use compounds that
exhibit high
therapeutic indices. The data obtained from these cell culture assays and
animal studies
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can be used in formulating a range of dosage for use in humans. The dosage of
such
compounds lies typically within a range of circulating concentrations that
include the
ED50 with little or no toxicity. The dosage may vary within this range
depending upon
the dosage form employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by the
individual
physician in view of the patient's condition.
[0268] Dosage amount and interval may be adjusted individually to provide
plasma levels of the active moiety which are sufficient to maintain the kinase
modulating
effects, or minimal effective concentration (MEC). The MEC will vary for each
compound but can be estimated from in vitro data; e.g., the concentration
necessary to
achieve 50-90% inhibition of the kinase. Dosages necessary to achieve the MEC
will
depend on individual characteristics and route of administration. However,
HPLC assays
or bioassays can be used to determine plasma concentrations.
[0269] Dosage intervals can also be determined using MEC value. Compounds
should be administered using a regimen that maintains plasma levels above the
MEC for
10-90% of the time, for example from about 30 to about 90%, such as from about
50 to
about 90%.
[0270] In cases of local administration or selective uptake, the effective
local
concentration of the drug may not be related to plasma concentration. The
amount of
composition administered will, of course, be dependent on the subject being
treated, on
the subject's weight, the severity of the affliction, the manner of
administration and the
judgment of the prescribing physician.
[0271] A suitable composition may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms containing
the active
ingredient. The pack may for instance include metal or plastic foil, such as a
blister pack.
The pack or dispenser device may be accompanied by instructions for
administration.
The pack or dispenser may also be accompanied with a notice associated with
the
container in a form prescribed by a governmental agency regulating the
manufacture,
use, or sale of pharmaceuticals, which notice is reflective of approval by the
agency of
the form of the compound for human or veterinary administration. Such notice,
for
example, may be the labelling approved by the U. S. Food and Drug
Administration or
other government agency for prescription drugs, or the approved product
insert.
[0272] As already noted above, in some embodiments a method according to the
invention includes determining, including monitoring, the migration of immune
cells
expressing CD45 and CD49d. In some of these embodiments the subject of whom
the
sample has been obtained is undergoing treatment with a VLA-4 blocking agent.
Generally, CD45d is expressed on all leukocytes, and CD49d is expressed on T
cells, B
cells, monocytes, eosinophils and basophils. In some embodiments the immune
cell to be
tested is T cell. In one embodiment the T cell is a CD4 ' T cell. In one
embodiment the T
cell is a CD8 T cell.
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[0273] Migratory capacity of immune cells is a prerequisite for immune
reactions. A respective method of the invention which can be used to evaluate
a subject's
immune competence and susceptibility to PML. Any technique that is suitable
for
determining the migratory capacity of an immune cell can be used. This can be
done
using any known techniques in the art. In some embodiments a chemotaxis assays
is
employed. Such assays are based on the functional migration of cells induced
by a
compound, and can be used to assess the binding and/or chemoattractant effect
of e.g.
ligands, inhibitors, or promoters. The use of an in vitro assay is illustrated
in the
Examples and also disclosed in U.S. Pat. No. 5,514,555. In some embodiment
chemotaxis assay determines the migration across endothelium into a collagen
gel
(described in Kavanaugh et al, J. Immunol (1991) 146, 4149-4156). Such assay
may
involve the use of a transwell-based set-up. In some embodiments a
chemoattractant is
dissolved in the medium on one side of a migratory barrier such as a polymeric
gel. On
the other side of the migratory barrier the cells of the sample from the
patient are
positioned. The migratory barrier is porous to a certain extent so that the
cells of interest
such as T cells are able to migrate through the same. The pores of the porous
migratory
barrier further allow the passage of chemoattractant molecules, so that a
diffusion
gradient forms, which can be detected by the cells of interest. As a result
the cells are
attracted to migrate across the migratory barrier. Typically the cells are
allowed to
migrate in the experimental setup for a certain, e.g. predetermined, period of
time,
whereafter the number of migrated cells and/or the migration distance is being
determined. For this purpose the migratory barrier may be analyzed under a
microscope.
The cells may also be stained before starting the chemotaxis assay and their
position may
be determined according to the signals obtained from the stain.
[0274] In one embodiment, the migratory capacity is compared to that obtained
from the same patient prior to the treatment of VLA-4 blocking agent. The
value
obtained can be set to 100%. After treatment is initiated, a drop in immune
cell migration
can be observed and compared. The migration at a given time point can be
characterized
by an average (mean) value coupled with a standard deviation value. Cell
migration in a
subject may be considered different when it is more than one standard
deviation different
from the average value (supra). The reference value may be defined as the mean
minus 1
standard deviation. When the difference falls below the reference value, it
may be
indicative of an increased risk for PML occurrence in the subject. The above
said with
regard to a threshold value in this regard applies mutatis mutandis.
[0275] As an illustrative example the following table provides an exemplary
reference level for immune cell migration that may be used. In this instance,
migration of
CD3 ' T cells over endothelium has been monitored over a period of time.
Month % of migrated CD3-' T cells in reference level (mean %
relation to untreated patients (set to of migrated CD3 ' T cells
100%) (mean (standard deviation)) minus 1 standard
deviation)
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0 (before 100.0 (none)
treatment)
1 62.7 (27.5) 35.2
3 38.8 (7.0) 31.8
6 11.1 (11.3) 0
12 31.3 (22.3) 9.0
15-20 71.7 (38.9) 32.8
21-25 104.7 (61.8) 42.9
26-30 61.8 (36.9) 24.9
31-35 35.7 (22.7) 13.0
36-40 57.5 (25.7) 31.8
41-45 57.0 (22.6) 34.4
46-50 104.6 (48.8) 55.8
51-55 119.8 (45.6) 74.2
Table 3: Exemplary reference values for migration of CD3 ' T cells
[0276] Immune cells have a basic capacity to migrate over cellular barriers
and
permeable membranes. The inventors have found that a lack or reduced CD62L
expression is associated with a strongly reduced migratory capacity. A
migration assay
used in a method according to the present invention may involve the use of a
permeable
membrane. The membrane may be any membrane commonly used in the field, such as
polycarbonate (PC), polyester (PET), and collagen-coated
polytetrafluoroethylene
(PTFE) membrane, which are available commercially (for example Transwell
membrane). A migration assay over a blank permeable membrane, i.e. without
cells, may
be used for such assessment. In some embodiments a migratory assay involves
the use of
cells. Cells such as endothelial cell or cell lines are within the scope of
the present
invention. Exemplary cells are -end cell line, c-end cell line, Human
umbilical vein
endothelial cell (HUVEC) cell line. In some embodiments primary human choroid
plexus-derived epithelial cells are employed. In some embodiments primary
human brain
microvasculary endothelial cells are used.
[0277] In addition, further known biomarkers can be optionally used as
secondary markers in the context of the present invention to assist the
assessment of the
susceptibility to PML of a patient receiving VLA-4 blocking agents. Such
markers
include the treatment duration, pretreatment with immunosuppressants, as well
as the
serum-positivity of JCV antibodies. In another aspect, the present invention
provides a
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method of treating a patient comprising administering the patient with VLA-4
blocking
agents, and measuring or detecting the level of expression of CD62L from the T
cell, and
stopping or continuing the administration based on the level of expression. As
described
earlier, the level of CD62L expression can be used to indicate risk or
occurrence of PML.
Thus, if the level indicates that there is an elevated risk of PML, the
treatment should be
stopped; otherwise, the treatment may continue.
[0278] VLA-4 blocking agent can be used to treat a number of diseases and
disorders, including multiple sclerosis, Crohn's disease, rheumatoid
arthritis, meningitis,
neuromyelitis optica, neurosarcoidosis, CNS vasculitis, chronic inflammatory
demyelinating polyradiculoneuropathy (CIDP), encephalitis, transverse
myelitis, tissue
or organ graft rejection or graft-versus-host disease, chronic renal disease,
CNS injury,
e.g., stroke or spinal cord injury; chronic renal disease; allergy, e.g.,
allergic asthma; type
1 diabetes; inflammatory bowel disorders, e.g., ulcerative colitis; myasthenia
gravis;
fibromyalgia; arthritic disorders, e.g., rheumatoid arthritis, psoriatic
arthritis;
inflammatory/immune skin disorders, e.g., psoriasis, vitiligo, dermatitis,
lichen planus;
systemic lupus erythematosus; Sjogren's Syndrome; hematological cancers, e.g.,
multiple
myeloma, leukemia, lymphoma; solid cancers, e.g., sarcomas or carcinomas,
e.g., of the
lung, breast, prostate, brain; and fibrotic disorders, e.g., pulmonary
fibrosis,
myelofibrosis, liver cirrhosis, mesangial proliferative glomerulonephritis,
crescentic
glomerulonephritis, diabetic nephropathy, and renal interstitial fibrosis.Any
disease or
pathological condition which has been treated or is known to be treatable by
the blocking
agent is part of the present invention. Accordingly, the treatment comprises
administering a therapeutically effective amount of VLA-4 blocking agents.
Patients may
be first subjected to prior screening to determine whether the treatment would
be
suitable. For example, the screening may be based on the patient history,
previous use of
immunosuppressant, Expanded Disability Status Scale (EDSS) in case of multiple
sclerosis patients, anti-JCV antibody status (JCV antibody seropositivity),
MRI imaging
studies, pre-infusion checklist for continuously worsening neurological
symptoms, and
other criteria commonly used.
[0279] Patient undergoing VLA-4 blocking agent treatment is tested to
determine the expression level of the biomarkers as disclosed herein. Samples
from the
patient are collected and analyzed. Preferably, they are sent to a central
testing facility to
ensure that the analysis of phenotype and function can be carried out under
standardized
conditions. In a preferred embodiment, the samples are taken at month 1, every
3 months
until the first year, and then every 6 months thereafter. In one embodiment,
the treatment
should be stopped if there is a lack of CD62L expression on T cells since this
indicates
an increased risk occurrence of PML.
[0280] The determination whether to stop the treatment or not can be based on
comparing the level of expression with a reference. As disclosed herein, the
reference
may be derived from one or more patients known to have suffered from PML or
other
complications, or one or more patients known to have not suffered from PML or
other
complications. For example, reference value or level can be gathered from
control
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patients. Expression levels of the biomarkers from the control patients using
any suitable
method may be recorded over a period of time, such as over 2-3 years. Average
expression levels, standard deviation, and relative standard deviation at
given times can
be calculated for the control patients to determine a range of expression
levels associated
with the control patients. When test result from a patient to be evaluated is
collected, it
will be compared to the reference value. Statistical differences between the
test result and
the reference will be determined to identify significant variances In between.
[0281] may be provided in the Based on CD62L expression, the physician is able
assess whether to continue, restart or stop the treatment of VLA-4 blocking
agents. The
information provides significant information to the physician regarding the
risk
associated with the treatment, so that informed benefit-risk decisions can be
made
accordingly.
[0282] It is to be understood that the methods of the present invention cannot
provide a 100 % prediction whether or not a patient will develop PML or other
complications, since individual factors such as other preexisting condition,
general
health, drug interaction and the like may have an influence as to whether or
not a patient
will be susceptible to PML
[0283] In addition, if a CD62L expression level is detected that indicates
that a
subject is at an elevated risk of PML occurrence, diagnosis with regard to PML
may be
intensified. As further explained below, MRI imaging may be employed to
identify any
area of demyelination. Further, cerebrospinal fluid may be analyzed for the
presence of
JCV DNA, or blood or a brain sample may be analyzed with regard to the
presence of
TNFR1 or TNF-a. If any of these diagnostic measures have previously been
carried out
on the subject, including carried out on a regular basis, if on the basis of
CD62L
expression levels, a subject is found to be at an elevated risk of developing
PML, one or
more such means of diagnosing PML may be carried out on a regular basis,
including on
a more frequent basis than previously done. As an illustrative example, it may
be
decided by the physician that every three months MRI imaging is carried out on
the
subject's brain.
[0284] In some embodiments the treatment includes administering a VLA-4
blocking agent to the subject. The method further includes measuring or
detecting the
level of expression of CD62L on T cells of the subject. Based on the level of
expression
of CD62L on the subject's T cells the administration of the VLA-4 blocking
agent is
stopped or continued. A threshold value may be used as a decision threshold
(supra). If a
CD62L expression level is detected that indicates that there is no elevated
risk of PML
occurrence, the administration of the VLA-4 blocking agent may be continued.
If a
CD62L expression level is detected that indicates that there is an elevated
risk of PML
occurrence, the administration of the VLA-4 blocking agent should be stopped.
In some
embodiments measures are taken to remove the VLA-4 blocking agent from the
subject's plasma if an elevated risk of PML occurrence has been determined. As
explained above plasma exchange or immunoadsorption may be carried out in this
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regard. In some embodiments stopping the administration of a VLA-4 blocking
agent
means that therapy with a VLA-4 blocking agent is entirely stopped, i.e. no
alternative
VLA-4 blocking agent is administered instead of the previously administered
VLA-4
blocking agent. Entirely ending or adjourning therapy with a VLA-4 blocking
agent may
assist reconstitution of the subject's immune surveillance. It is further
noted in this
regard that a subject suffering from MS and under therapy with a VLA-4
blocking agent
is often a subject that/who did not respond to a first-line therapy such as
interferon-13 or
glatiramer acetate. Beginning such a therapy as a substitute of VLA-4 blocking
agent
therapy may therefore only have a low chance of improving the subject's
condition.
[0285] In another aspect the present invention provides a kit. Such kit
includes
an assay kit for detecting one or more biomarkers as provided in the present
invention.
Means for detecting a biomarker are known in the art, and include, for
example, the use
of a capture probe such as an immunoglobulin or a proteinaceous binding
molecule with
immunoglobulin-like functions, which optionally is detectably labeled. As
explained
above the capture probe may be used together with a detection agent that binds
to the
biomarker and/or the capture probe. A capture probe may be, for example and
not by
limitation, an antibody, a portion of an antibody such as a Fab or Fab2
fragment, a single
chain antibody, a receptor for the biomarker or a portion thereof or a ligand
for the
biomarker or a portion thereof. Likewise, a detection agent may be, for
example and not
by limitation, an antibody, a portion of an antibody such as a Fab or Fab2
fragment, a
single chain antibody, a receptor for the biomarker or capture agent or a
portion thereof
or a ligand for the biomarker or capture probe or a portion thereof. The
capture probe
and/or detection agent may be detectably labeled using a radioactive label, a
fluorescent
label, a chemical label, an oligonucleotide label, an enzymatic label, or a
protein label
(e.g. a fluorescent protein such as Green Fluorescent Protein). It is to be
understood that
the method may also include any molecules which can be used to indirectly
indicate the
level of the biomarkers. A "capture probe" may be a molecule that binds an
mRNA or
protein through an interaction that is sufficient to permit the binding to the
mRNA or
protein.
[0286] In one embodiment the kit may include a CD62L specific capture probe,
and optionally a LFA-1 capture probe. The kit may further include a CD3
specific
capture probe. In some embodiments the kit may further include a CD4 specific
capture
probe and/or a CD8 specific capture probe. In some embodiments the kit may
include a
multi-specific capture probe directed to CD3, CD62L and LFA-1, optionally
together
with a detection agent. In one embodiment the kit includes components for
setting up an
assay for CD3 and CD62L. In some embodiments the kit includes an
immunoglobulin or
a proteinaceous binding molecule with immunoglobulin-like functions, or any
other
capture probe directed to the protein or mRNA of CD11a, and a capture probe
directed to
the protein or mRNA of CD18. Such a kit may also include a capture probe
directed to
the protein or mRNA of Runx3.
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[0287] A respective kit may furthermore include means for immobilising the
capture probe to a surface. As explained above, a nucleic acid capture probe
included in
the kit may have a moiety that allows for, or facilitates, an immobilisation
on a surface.
[0288] The kit may further include instructions and/or imprint indicating that
a
patient is to be stratified by a method described herein; and/or instructions
regarding how
to carry out a method as defined herein. It may also include positive and/or
negative
controls which allow a comparison to the control. The kit shall enable the
assessment of
a patient's treatment progress and the susceptibility to PML. A respective kit
may be used
to carry out a method according to the present invention. It may include one
or more
devices for accommodating the above components before, while carrying out a
method
of the invention, and thereafter.
[0289] As indicated above, any number of steps of a method according to the
invention, including the entire method, may be performed in an automated way ¨
also
repeatedly, using for instance commercially available robots. As an
illustrative example,
the method may be an in vitro screening method, for example carried out in
multiple-well
microplates (e.g. conventional 48-, 96-, 384- or 1536 well plates) using
automated work
stations. The method may also be carried out using a kit of parts, for
instance designed
for performing the present method.
[0290] The listing or discussion of a previously published document in this
specification should not necessarily be taken as an acknowledgement that the
document
is part of the state of the art or is common general knowledge.
[0291] The invention illustratively described herein may suitably be practiced
in
the absence of any element or elements, limitation or limitations, not
specifically
disclosed herein. Thus, for example, the terms "comprising", "including,"
containing",
etc. shall be read expansively and without limitation. Singular forms such as
"a", "an" or
"the" include plural references unless the context clearly indicates
otherwise. Unless
otherwise indicated, the term "at least" preceding a series of elements is to
be understood
to refer to every element in the series. The terms "at least one" and "at
least one of'
include for example, one, two, three, four, or five or more elements. Slight
variations
above and below the stated ranges can be used to achieve substantially the
same results
as values within the ranges. Also, unless indicated otherwise, the disclosure
of the ranges
is intended as a continuous range including every value between the minimum
and
maximum values.In a further aspect, the invention also relates to the use of a
kit
comprising CD62L and optionally LFA-1 binding assay to determine the immune
competence of a patient undergoing a treatment comprising VLA-4 blocking
agent. The
binding assay preferably comprises a CD62L and optionally a LFA-1 capture
probe as
described earlier. The kit enables the assessment of the risk for PML during
the course of
the treatment. Put it differently, CD62L can be seen as dynamic biomarkers
which can
assist the physician to determine whether to stop, continue, or resume the
treatment of
VLA-4 blocking agent, or to make any other suitable adjustment of the
treatment
regimen.
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[0292] The practice of the present invention will employ conventional
techniques of molecular biology, microbiology, and immunology, which are
within the
skill of those working in the art. Such techniques are explained fully in the
literature.
Examples of particularly suitable texts for consultation include the
following: Sambrook
et al. (1989) Molecular Cloning; A Laboratory Manual (2d ed.); D. N Glover,
ed. (1985)
DNA Cloning, Volumes I and II; M. J. Gait, ed. (1984) Oligonucleotide
Synthesis; B. D.
Hames & S J. Higgins, eds. (1984) Nucleic Acid Hybridization; B. D. Hames & S.
J.
Higgins, eds. (1984) Transcription and Translation; R. I. Freshney, ed. (1986)
Animal
Cell Culture; Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical
Methods in Cell and Molecular Biology (Academic Press, London); Scopes (1987)
Protein Purification: Principles and Practice (2d ed.; Springer Verlag, N.Y.);
and D. M.
Weir and C. C. Blackwell, eds. (1986) Handbook of Experimental Immunology,
Volumes I-IV.
[0293] Additionally, the terms and expressions employed herein have been used
as terms of description and not of limitation, and there is no intention in
the use of such
terms and expressions of excluding any equivalents of the features shown and
described
or portions thereof, but it is recognized that various modifications are
possible within the
scope of the invention claimed. Thus, it should be understood that although
the present
invention has been specifically disclosed by exemplary embodiments and
optional
features, modification and variation of the inventions embodied therein herein
disclosed
may be resorted to by those skilled in the art, and that such modifications
and variations
are considered to be within the scope of this invention.
[0294] The invention has been described broadly and generically herein. Each
of
the narrower species and subgeneric groupings falling within the generic
disclosure also
form part of the invention. This includes the generic description of the
invention with a
proviso or negative limitation removing any subject matter from the genus,
regardless of
whether or not the excised material is specifically recited herein.
[0295] Other embodiments are within the appending claims. In addition, where
features or aspects of the invention are described in terms of Markush groups,
those
skilled in the art will recognize that the invention is also thereby described
in terms of
any individual member or subgroup of members of the Markush group.
[0296] In order that the invention may be readily understood and put into
practical effect, particular embodiments will now be described by way of the
following
non-limiting examples.
EXAMPLES
[0297] The examples illustrate techniques that can be used in methods
according
to the invention as well as exemplary embodiments of determining the level of
T cells
that express L-CD62L and/or LFA-1. Studies in recent years have come up with
three
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statistical observations of predicting an MS patient's overall risk to later
develop PML
when treated with Natalizumab, but there is no possibility yet to measure an
individual's
PML risk. This, however, is urgently needed to facilitate the treatment
decisions of
clinicians and patients alike, as many patients opt for continuing treatment
with
Natalizumab even when their statistical risk to develop PML rises up to
roughly 1:120
with all three risk factors present. As compromised immune surveillance has
long been a
hypothesis to explain the occurrence of an opportunistic infection, the
inventors designed
a study to include a variety of adhesion molecules on the surface of immune
cell
subpopulations. Patients analysed included inter alia MS patients under long-
term
therapy with Natalizumab, MS patients before escalation to Natalizumab, but
with
diverse prior immunemodulatory treatments such as e.g. Glatiramer Acetate,
Interferon-
p, Azathioprine, or Methotrexate.
[0298] The obtained data show that a surface molecule was strongly
downregulated on T cells of patients who either would later develop PML. This
molecule
was CD62L, a member of the selectin family. In addition, LFA-1 was likewise
strongly
downregulated on T cells of MS patients under Natalizumab therapy before
occurrence
of PML. These markers are therefore risk predictors, because their expression
pattern
during Natalizumab therapy differed.
[0299] As the expression of L-Selectin was usually higher on CD4 ' T cells
(because in contrast to CD8 T cells, there are no CD4 'CD62L-CD45RA' cells),
it may
in some cases be advantageous to use CD4 ' T cells to determine risk when
using L-
Selectin.
Subjects treated with Natalizumab
[0300] The status and longitudinal development, as well as function of major
peripheral and CSF immune populations in patients under long-term treatment
with
Natalizumab were assessed. Focus of the following experiments underlying these
examples was finding changes in the immune status of patients to possibly
predict the
occurrence of PML by assessing the impact of Natalizumab on T-cell function in
combining immune phenotyping with functional in vitro and ex vivo assays.
[0301] Natalizumab, a humanized IgG4 antibody against the cc-chain of VLA-4
(a4, CD49d), has been approved for the treatment of active relapsing-remitting
Multiple
Sclerosis (RRMS) since 2006. Long-term treatment with Natalizumab is
associated with
severe side effects, above all the development of progressive multifocal
leukencephalopathy (PML). In addition to duration of treatment, previous
immunosuppressive therapy (Panzara, M.A., et al. Multiple Sclerosis (2009) 15,
9, S132-
S133) as well as the presence of JC virus, as ascertained by the presence of
anti-JCV
antibodies in serum, contribute to the risk of developing Natalizumab-
associated PML
(Bloomgren, G., et al. The New England Journal of Medicine (2012) 366, 20,
1870-
1880). When all these risk factors are present, the statistical risk of PML
can be as high
as 1:120 (Clifford, D. B., et al. Lancet Neurology (2010) 9, 4, 438-446;
Bloomgren et al.,
2012, supra). While it is still unclear why and how (long-term) treatment with
anti-
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CD49d contributes to the development of PML (Tan and Koralnik, 2010, supra), a
multifactorial scenario is likely, including impaired immune surveillance and
active JC
virus replication (Schwab, N., et al. Multiple sclerosis [Houndmills,
Basingstoke,
England] (2012) 18, 3, 335-344; Schwab, N., et al. Neurology (2012) 78, 7, 458-
467).
[0302] 114 patients with the diagnosis of clinically definite active RRMS
according to the 2005 revised McDonald diagnostic criteria (Polman, C.H. et
al., Ann
Neurol (2005) 58, 840-846) were enrolled in this study. 67 MS patients had
continuously
been treated with Natalizumab for 18-66 months, 21 MS patients received
baseline
immune-modulatory treatments (Interferons, Glatiramer acetate) and 26 MS
patients
were untreated and clinically as well as MRI-stable (cf. also Table 1, supra,
for 22 of
these patients). Age and sex-matched healthy donors (HD) with no previous
history of
any neurological or immune-mediated diseases served as controls. Furthermore,
samples
were available from different therapy-associated PML conditions: Natalizumab-
associated (n=13), Rituximab-associated (n=1) and Efalizumab-associated (n=1).
Six
cases of HIV-associated PML served as additional controls. In 6 of the 13
Natalizumab-
associated PML cases pre-PML samples were available (Fig. 15). The Table
depicts
average + standard deviation (if applicable).
[0303] The study was approved by the local ethics committee (Ethik-
Kommission der medizinischen Fakultat der Universitat Wiirzburg, registration
number
155/06; Ethik-Kommission der Arztekammer Westfalen-Lippe und der Medizinischen
Fakultat der Wesfalischen Wilhelms-Universitat, registration number: 2010-245-
f-S) and
informed written consent was obtained from all participants. This study was
performed
according to the Declaration of Helsinki.
[0304] Data shown in Fig. 1D, Fig. 7, Fig. 8 and Fig. 9 are based on a smaller
group of patients. 52 patients with the diagnosis of clinically definite
active RRMS
according to the 2005 revised McDonald diagnostic criteria were enrolled.
Analyzed MS
patients had been treated continuously with Natalizumab for 18-50 months and
were
stable by assessment of clinical and MRI parameters. 18 patients among this
cohort
underwent analysis of CSF in parallel to assessment of peripheral blood. 39
patients were
followed longitudinally from treatment initiation. Two patients developed PML
after 26
or 29 months, respectively. 45 age and sex-matched healthy donors (HD) with no
previous history of neurological or immune mediated diseases served as
controls.
Furthermore, 22 untreated MS patients served as controls (Table 1, supra).
PBMC from
patients suffering from PML (HIV (n=4), Natalizumab-associated (n=3),
Rituximab-
associated (n=1), Efalizumab-associated (n=1)) were used as additional
controls.
[0305] Peripheral blood (n=52) and cerebrospinal fluid (n=18) from patients
under Natalizumab therapy (>18 months) were analyzed using flow cytometry and
in
vitro transendothelial migration assays.
[0306] Data shown in Fig. 3 are based on a group of patients of yet different
size. 78 patients with the diagnosis of clinically definite active RRMS were
included.
Analyzed MS patients had been treated continuously with Natalizumab for 18 to
60
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months and were stable by assessment of clinical and MRI parameters. Five
patients
developed PML. In addition, samples were obtained from 30 patients with the
diagnosis
of clinically definite active RRMS before treatment with Natalizumab. 73 age
and sex-
matched healthy donors with no previous history of neurological or immune
mediated
diseases served as controls.
Isolation of PBMC and Flow Cytometric Analysis
[0307] Peripheral blood mononuclear cells (PBMC) freshly isolated from EDTA
blood were isolated by density gradient centrifugation using lymphocyte
separation
medium (PAA Laboratories, Pasching, Austria) as described previously in Schwab
et al J
Immunol. (2010) 184, 9, 5368-5374, incorporated herein by reference in its
entirety.
Flow cytometry analysis of CSF was performed as described in Schwab et al.
Multiple
Sclerosis (2009) 15, S275¨S275, incorporated herein by reference in its
entirety. Cells
were then typically cryopreserved in freezing medium (50% RPMI 40% FCS 10%
DMSO).
[0308] Ex vivo isolated, cultured or thawed cells were washed with FACSO-
buffer (phosphatebuffered saline (PBS) supplemented with 0.1% bovine serum
albumine
(BSA) and 0.1% NaN3) or staining buffer (phosphate-buffered saline (PBS)
supplemented with 0.1% bovine serum albumine (BSA) and 200 mM EDTA). Cells
were
subsequently stained with fluorescence-labeled monoclonal antibodies (Mab)
together
with blocking mouse IgG (Sigma-Aldrich, Hamburg, Germany) at 4 C for 30 mm or
at
room temperature for 15 mm. After washing once with staining buffer, cells
were
immediately measured on a FACSCalibur (BD Biosciences, Heidelberg, Germany)
and
GalliosTM Flow Cytometer (Beckman Coulter, Krefeld, Germany) and analyzed
using
FlowJo (Tree Star, Ashland, OR, USA) and Kaluza (Beckman Coulter) software. It
should be noted that the presence of CD62L on the cell surface tends to be
unstable, so
that the staining buffer cannot contain sodium azide and measurement needs to
take place
immediately after the staining procedure.
[0309] In particular, LFA-1 protein was stained for CD1 1 a, the a-chain of
LFA-
1. VLA-4 was stained for CD49d (the cc-chain of VLA-4), as CD49d is the
precise
molecule blocked by Natalizumab.
[0310] Monoclonal immunoglobulins used in these examples were anti-CD62L
(DREG-56, BioLegend), anti-CD3 (UCHT1, Beckman Coulter), anti-CD4 (13B8.2,
Beckman Coulter), anti-CD8 (B9.11, Beckman Coulter), anti-CD1 1 a (HIM, BD
Pharmingen), anti-CD14, (MoP9, BD Biosciences), anti-CD19 (HIB19, BD
Biosciences), anti-CD45 (J33, Beckman Coulter), anti-CD45RA (HI100, Beckman
Coulter), anti-CD56 (NCAM 16.2, BD Biosciences), anti-CD49d (9F10, Biolegend)
and
anti-CD197 (3D12, BD Biosciences).
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Immunohistochemistry
[0311] Retrospectively investigated were 2 chordoid plexus tissue samples
(autopsies) from 2 multiple sclerosis patients (both female, 31 and 72 years),
and 15
tissue samples from patients without neurological diseases (11 men, 4 women;
between
34 and 81 years, mean 60 years). The study was approved by the Ethics
Committee of
the University of Muenster. For histological analysis the paraffin embedded
tissue
samples were cut in 4 pm thick sections and stained with haematoxylin and
eosin (HE).
Immunohistochemistry for mouse anti-human CD3 (1 : 25) (Dako, Denmark) was
performed using an automated immunostainer (autostainer Link48, Dako) and the
avidin-
biotin technique. Steamer pretreatment (citrate buffer pH6.1) for better
antigen retrieval
was performed.
In vitro migration assays
[0312] Primary human brain microvascular endothelial cells (HBMEC) and
primary human choroid plexus epithelial cells (HCPEpiC) were purchased from
ScienCell Research Laboratories (San Diego, CA, USA). Cells were cultured on
filter
membrane of Transwells (3pm pore size; Corning, New York, USA) for three days
until
reaching confluence.
[0313] Transmigration assays were performed essentially as described in
Schneider-Hohendorf et al. Eur J Immunol. (2010) 40, 12, 3581-3590,
incorporated
herein by reference in its entirety. Briefly, PBMC in 100p1 of pre-warmed RPMI
medium
(RPMI, Penicilline/Streptamycine (1%), B27 supplement (2%) [Invitrogen,
Darmstadt,
Germany]) were added to the top of the HBMEC monolayers, and 600 pl of medium
were added to the outer chamber of the inserts. The cells were allowed to
migrate for six
hours in a humidified cell culture incubator at 37 C and 5% CO2. Absolute
counts of T
cells were measured with Flow-Count Fluorospheres following the manufacturer's
instructions (Beckman Coulter) to normalize the migration rates to
standardized bead
concentrations.
Statistical analysis
[0314] Statistical significance of differences between two groups was
determined using unpaired Student's t-test except for comparisons between
peripheral
blood and CSF of the same patient, where the paired Student's t-test was used.
Differences were considered statistically significant with p* values <0.05,
with p**<0.01
and p***<0.001. Software for statistical and correlation assessment was Prism
5
(GraphPad, La Jolla, CA, USA).
Changes in the composition of major immune subsets under long-term
Natalizumab therapy
[0315] Characterization of the major peripheral immune cell subpopulations in
patients under long-term treatment with Natalizumab (n=34, treatment > 18
months)
(Fig. 2). The percentage of CD4 ' T cells did not deviate significantly from
healthy
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donors and untreated MS patients. The CSF compartment of these patients (n=18)
was
characterized by reduced percentages of B cells and CD4-' T cells compared to
peripheral
blood. The CD4/CD8 ratio in the CSF was reduced to 0.54 (11.8:21.8),
indicating a
stronger effect of Natalizumab on CD4 than CD8 T cells (Fig. 4).
Impact of long-term Natalizumab treatment on T-cell function and phenotype
[0316] As published previously (Defer, G., et al., J Neurol Sci (2012) 314, 1-
2,
138-142; Harrer, A., et al., J Neuroimmunol (2011) 234, 1-2, 148-154),
treatment with
Natalizumab influenced the expression of CD49d on patients' peripheral CD4 ' T
cells
over time. However, it could be observed that after a decrease of surface
expression to a
minimum at month six of treatment, the CD49d levels surprisingly recovered. Of
note, it
could be shown that a patient, who developed antibodies against Natalizumab,
did not
downregulate CD49d on CD4 ' T cells, which might easily be used as an early
marker for
the detection of patients who will not benefit from Natalizumab due to
production of
antibodies, as previously suggested by (Defer et al., 2012, supra). Never-the-
less, CSF
flow cytometry showed that CD49d levels on CD4-' T cells were undetectable in
these
patients compared to their peripheral counterparts, independent of the
peripheral
recovery (Fig 2B), whereas it has been shown repeatedly that control MS
patients usually
show a strongly enhanced CD49d expression on CSF T cells when compared to the
periphery (data not shown and (Barrau, M.A. et al., J Neuroimmunol (2000) 111,
215-
223). Additionally, CSF CD4 ' T cells in patients under long-term treatment
were
characterized by missing expression of CD45RA and CCR7 (indicating an effector
memory phenotype). This stands in contrast to the central-memory-like
phenotype
(CD45RA-CCR7'), which has been published previously for MS patients (Kivisakk,
P.,
et al., Ann Neurol (2004) 55, 627-638). Similar results were obtained for CD8
' T cells
(data not shown). Effector memorycompartments (as determined by CCR7
expression) in
the periphery were not significantly affected by Natalizumab long-term therapy
(data not
shown)(Planas, R., et al., Eur J Immunol. (2011) doi: 10.1002/eji.201142108).
CSF is
generated in the choroid plexus (CP), which has also been shown in animal
models to be
the main entry site for leukocytes during CNS immune surveillance (Carrithers,
M.D., et
al., Brain (2000) 123 (Pt 6), 1092-1101) as well as inflammation (Reboldi, A.,
et al., Nat
Immunol (2009) 10, 514-523). The inventors could show that this route is a
possible
entry site for T cells in the human system during homeostatic as well as
pathological
conditions. In both MS tissue samples as well as in 7 out of 15 controls we
detected CD3
positive cells in the choroid plexus. The majority of T cells was located
perivascularly,
however we observed also single T cells in close proximity to the epithelium.
As
administration of Natalizumab is assumed to reduce CNS-invasion of leukocytes
by
inhibiting immune cell adhesion to endothelial cells of the blood-brain
barrier (BBB), it
was unexpected that quantitative comparison of individual migration through
primary
human brain-derived microvascular endothelium revealed a strong heterogeneity
among
Natalizumab-treated patients compared to healthy controls or untreated MS
patients (Fig.
5), even though all treated patients were considered clinically stable. In
contrast to this,
diapedesis through primary choroid plexus-derived epithelium (simulating the
blood-
CSF barrier) revealed a significant and homogeneous reduction in long-term
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Natalizumab-treated patients (Fig. 6). As the inventors had observed a
relation between
the expression of CD49d and treatment duration, they decided to analyze the
apparent
heterogeneity of transendothelial migration in relation to the months of
Natalizumab
treatment in more detail.
Longitudinal assessment of T-cell function under Natalizumab treatment:
Implications for the development of PML
[0317] Therapy-associated PML has developed as a significant challenge in a
number of medical specialties over the past several years (Vinhas de Souza,
M., et al.,
Clinical Pharmacology and Therapeutics (2012) 91, 4, 747-750). Natalizumab-
associated
PML has attracted considerable attention, since anti-CD49d treatment has been
associated with a particularly large number of PML cases in a population,
which is
traditionally not at risk. Three factors have been identified that can be used
as risk
stratification tools. Two, namely prior immunosuppressant use and duration of
therapy,
are based on statistical observations, while one, presence of anti-JCV
antibodies, is based
on a patient's specific biologic parameter. However, even JCV seropositivity
is relatively
non-specific, since it simply identifies patients who have had or currently
have a JCV
infection and therefore the theoretical possibility of developing PML, which
is JCV-
mediated (Panzara et al., 2009, supra; Clifford et al., 2010, supra; Gorelik,
L., et al.
Annals of Neurology (2010) 68, 3, 295-303; Bloomgren et al., 2012, supra). A
method to
measure an individual's biological response to treatment as a way to monitor
for PML
risk is urgently needed. We used the following groups of blood donors to
differentiate
between effects of MS, pre-treatments, and Natalizumab: 1) healthy controls,
2)
treatment-naive MS patients, 3) MS patients before treatment with Natalizumab
and 4)
MS patients under long-term therapy with Natalizumab (18-66 months). The
Natalizumab-treated subjects were recruited from five separate cohorts
(Wiirzburg,
Munster, Osnabruck (Germany), French Cohort Study (France) and Brascia
(Italy)). In
part among these five cohorts, we had access to samples from 13 PML patients.
Importantly, six of these patients had given blood before the diagnosis of PML
(19, 26, 4,
15, 21, 20 months before PML diagnosis). As additional controls, we analyzed
samples
from non-Natalizumab patients who developed PML (both therapy-associated and
HIV-
associated; see study design and Table 1). Surprisingly, our results showed
that the
percentage of CD62L expressing cells was consitently much lower (by more than
tenfold) on CD4 T cells of patients who would later on develop PML with a mean
of
3.3% compared with a mean of 46.6% from non-PML Natalizumab patients (Fig. lA
and
Fig. 15 for individual dot plots).
[0318] Furthermore, samples from patients suffering from acute PML also
showed a reduction or lack of CD62L expression, indicating a persistent
dysregulation at
least up to the point of PML diagnosis. CD62L expression showed a more diverse
pattern
in PML patients post diagnosis, perhaps due to the acute treatments
administered for
management of the PML. After PML (recovery phase, post immune reconstitution
syndrome, IRIS), the percentage of CD4 ' cells expressing CD62L returned to a
more
normal range (45.4%) (Fig. 1B). Surface expression of CD62L on CD4 ' T cells
was
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higher than on CD8 T cells. Therefore, the detection of CD62L levels on CD4 '
T cells
allowed for the most accurate discrimination of patients who eventually
developed PML
(data not shown). Of note, the present inventors found that using the
percentage of
positive cells against the isotype (in contrast to the MFI) gave the most
reproducible
results on different flow cytometers. The detailed gating is sketched in Fig.
15.
[0319] Expression levels of CD62L and LFA-1 were followed longitudinally in
39 Natalizumab patients in relation to transendothelial migration. Notably,
levels of
peripheral LFA-1 (Fig. 7) and CD62L (Fig. 8) showed a pronounced decrease
within the
first months, with a minimum at 6 months of therapy, followed by a subsequent
gradual
recovery. Functionally, this shift (reduced levels of CD49d, LFA-1, and CD62L)
lead to
a pronounced reduction of T-cell migration until 6 months of therapy and a
subsequent
recovery (Fig. 9). Between months 3 and 12 of treatment, transendothelial
migration of T
cells in vitro is severely reduced, which coincided with the reduced
expression of CD62L
and LFA-1.
[0320] Two patients in the cohort on which the data of Fig. 1D, Fig. 7 and
Fig. 8
are based developed PML after 26 (Fig. 9, grey circles) and 29 (Fig. 9, white
circles)
months of therapy. Analysis of these patients' samples (time point 0 was not
available)
revealed that, in contrast to the normal development, levels of LFA-1 on CD4 '
T cells
further decreased after 12 months of therapy instead of the expected recovery
(Fig.7).
Additionally, CD62L expression was completely absent during the investigated
time
frame for one of the patient who later developed PML (Fig. 8) and the
migration of T
cells was already very low at month 1. Migratory function did not recover over
time (Fig.
9). Notably, analysis of one of these patients more than one year after PML
revealed a
restored transendothelial migration/CD1la expression with poor recovery of
CD62L
expression(.
[0321] Compared to the control patients (patients who did not develop PML),
PML patients showed a lack of LFA-1 recovery, (Fig. 7), a lack or reduced of
CD62L
expression and a lack of CD62L recovery (Fig. 8), and reduced transendothelial
migration (Fig. 9).
[0322] Patients suffering from PML (n=8) associated with HIV infection or
treatment with monoclonal antibodies (Natalizumab, Rituximab, Efalizumab)
showed a
similar lack of CD62L expression on the surface of CD4 ' T cells at the
beginning of their
PML. This was again not associated with a shift towards effector memory T
cells as
delineated by CD45RA/CCR7 stainings (data not shown).
[0323] Perhaps importantly, the effector-memory distribution (assessed by
CCR7) (Schwab et al., Multiple Sclerosis, 2012, supra; Sottini, A., et al.
PLoS ONE
(2012) 7, 4, e34493) of two of these patients was also altered, whereas the
other four
were comparable to HDs (data not shown). This may define a group with inherent
risk of
PML development under specific conditions.
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[0324] While more research is needed, the inventors' results suggest a
possible
treatment paradigm where, after more than 18 Natalizumab infusions (months of
therapy), the percentage of CD62L positive CD4 ' cells is assessed. If the
CD62L level
drops below a defined threshold, which in this study could be set to
approximately 25%,
(Fig. 1A, dotted line: defined as two times the standard deviation (SD) from
the mean
(m) of the control cohort (mean=46.6; SD=11.1; threshold=24.5)) an early re-
assessment
(e.g. one month later) of the percentage of CD62L expressing T cells may be
advisable.
Continuous lack of CD62L could indicate a higher risk of PML and warrant
either very
close clinical monitoring or a potential change in treatment regimens
(Natalizumab
cessation). As acute PML appears to exert variable, but not well understood
effects on
the immune system, CD62L should not be used as a method of PML diagnosis per
se, but
rather as a prospective risk factor for developing PML in the future.
[0325] Taken together, the present cell-based assay for PML risk prediction
may
provide an immensely valuable tool for patients and practitioners in the field
of MS
treatment, albeit it needs to be further validated in larger, multicenter
cohorts, as well as
using more patient samples collected before development of PML.
Real time PCR analysis
[0326] RNA isolation was performed using Trizol0 (Invitrogen, Karlsruhe,
Germany) following the manufacturer's instructions. mRNA was transcribed using
random hexamers and MuLV reverse transcriptase (all reagents supplied by
Applied
Biosystems, Foster City, USA). Gene expression assays for the detection and
quantification of CD11a, Runx3 and CD62L and the housekeeping gene hS18 were
purchased from Applied Biosystems and used according to the manufacturer's
protocol.
The Applied Biosystems Step-One Plus real-time PCR system was used, all
samples
were run in duplicates and each run contained several controls (healthy donor
samples,
wells without cDNA). There were no significant differences in cycle threshold
neither
within nor between the experiments. Quantification of gene expression was
performed by
comparing the amplification efficiencies of targets and housekeeping gene. All
samples
were normalized to hS18. Therefore, a lower CT value equals a higher
expression of
mRNA of the specific target. Figures 10-12 show the relative quantification of
CD1 1 a,
Runx3, and CD62L as compared to 11518 on thawed PBMC from MS patients before
(month 0) and in the time course of therapy (months 1, 3, 6, 12, 15-20, 21-25,
26-30, 31-
40, 41-50; n=27 patients) as assessed by real-time PCR.
[0327] Long-term treatment with Natalizumab leads to changes in the peripheral
immune subset distribution, which is in accordance to previous reports
(increased
numbers of peripheral B cells, attributed to the recruitment of precursor B
cells
(Krumbholz, M., et al., Neurology (2008) 71, 1350-1354) and decreased numbers
of
peripheral CD14 ' monocytes (Skarica, M., et al., J Neuroimmunol (2011) 235, 1-
2, 70-
76). The increase in peripheral CD8 T cells with no significant changes in the
CD4
compartment might possibly contribute to the reversed CD4/CD8 ratio in the
CSF, as
observed in the cohort of these Examples and previously published (Stiive, 0,
et al., Arch
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89
Neurol (2006) 63, 1383-1387). Not mutually exclusive, CD4 cells might also
undergo
apoptosis upon encountering the antibody for a prolonged period of time, which
has been
published for short-term exposure in vitro (Kivisakk, P., et al., Neurology
(2009) 72,
1922-1930). The alterations in CD62L and LFA-1 expression on T cells, which
have
previously been shown for their CD34 ' stem cells (Jing, D., et al., Bone
Marrow
Transplantation (2010) 45, 1489-1496), might also be due to the co-
localization of
CD49d with CD62L on cell surface microvilli (Wedepohl, S., et al., Eur J Cell
Biol.
(2012) 91, 4, 257-264). In contrast to CD62L, LFA-1 is solely expressed on the
planar
cell body (ibid.), suggesting that the expression of LFA-1 is regulated on the
gene
expression level, as the connection between CD49d and LFA-1 has been shown in
the
inverted setting, where the blockade of CD1 1 a increased the percentage of
CD49d T
cells (Harper, E.G., et al., J Invest Dermatol (2008) 128, 1173-1181).
[0328] LFA-1 and CD62L have previously been used together with CD45RA as
markers to distinguish naïve, central-memory, and effector-memory T cells
(Maldonado,
A., et al., Arthritis Res Ther (2003) 5, R91-R96; Okumura, M., et al., J
Immunol (1993)
150, 429-437). These subpopulations differ in their functional tasks with
central-memory
cells conferring immunity against viruses and cancer cells and effector-memory
cells
producing cytokines like IFN-y and IL-4 (reviewed in (Wherry, E., et al., Nat
Immunol.
(2003) 4, 3, 225-234). The CSF of MS patients has been shown to mainly consist
of
central memory cells (Giunti, D., et al., J Leukoc Biol (2003) 73, 584-590;
Kivisakk et
al., 2004, supra). This population is known to be involved in immune-mediated
CNS
damage during EAE (Grewal, I.S., et al., Immunity (2001) 14, 291-302) invading
the
CNS via the choroid plexus (Reboldi, A., et al., Nat Immunol (2009) 10, 514-
523). The
CSF of patients under long-term treatment with Natalizumab, however, almost
exclusively contains effector-memory-like T cells. Furthermore,
transepithelial migration
of long-term treated Natalizumab patients is permanently reduced while
transendothelial
migration recovers during long-term therapy. The choroid plexus divides blood
and CSF
consisting of two barriers, one endothelial barrier on the blood side and one
epithelial on
the CSF side (Engelhardt, B., et al., Microsc Res Tech (2001) 52, 112-129) and
reviewed
by (Wilson, E.H., et al., J Clin Invest (2010) 120, 1368-1379). In line with
previous
findings in the murine system, showing that CD49d is mandatory for adhesion to
the
epithelial-, but not to the endothelial barrier (Steffen, B.J., et al., Am J
Pathol (1996) 148,
6, 1819-1838) of the choroid plexus, it is conceivable that Natalizumab
efficiently
impairs this route to the CSF, resulting in a low cell count in the CSF of
patients and the
clinical anti-inflammatory effects. Immune surveillance, which can be
accomplished
using alternative routes e.g. via the subarachnoid space or directly through
the blood
brain barrier (reviewed by Hickey, W.F., Semin Immunol (1999) 11, 125-137),
should
still be functional in patients under long-term treatment with Natalizumab, as
they only
require crossing an endothelial barrier. In line with this, the T cells in the
CSF of
Natalizumab patients do not express CD49d, indicating that these cells did not
use the
choroid plexus as entry site into the CNS. It was shown very recently that
Th17 cells in
EAE migrate into the spinal cord independently of a4 integrin, whereas Thl
cells, which
are supposed to be mainly responsiblefor MS pathology, use a4 integrin for the
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migration into the brain (Rothhammer, V., et al., J Exp Med. (2011) 21, 208,
12, 2465-
2476). The invasion of these putatively pathogenic Thl cells would therefore
be inhibited
by Natalizumab.
[0329] Between months 3 and 12 of treatment, transendothelial migration of T
cells in vitro is severely reduced. This coincides with a reduced expression
of LFA-1 and
CD62L, both being molecules imperative for endothelial migration (reviewed by
(Ransohoff et al., Nat Rev Immunol. (2003) 3, 7, 569-581). Interestingly, this
fits to
previously published data, showing peaking JCV-, but also Epstein-Bar-,
Cytomegalo-
and MOBP-specific T-cell responses in the same time frame indicating that the
majority
of primed effector T cells are efficiently trapped in the periphery (Jilek,S.,
et al., Lancet
Neurol. (2010) 9, 3, 264-272). As a side note, the observed modulation of LFA-
1 should
have major implications for T-cell function besides migration, such as
formation of the
immunological synapse together with CD49d, cytotoxicity and antigen-specific
restimulation (Mittelbrunn, M., et al., Proc Nat! Acad Sci U.S.A. (2004) 27,
101(30):11058-63; Rutigliano, J.A., et al., 2004, J Virol. (2004) 78, 6, 3014-
3023;
Yarovinsky, T.O., et al., Am J Respir Cell Mol Biol. (2003) 28, 5, 607-615).
Admittedly,
the applied migration paradigms can only partly reflect the in vivo situation,
as especially
the inflammatory milieu at stages of a possible MS relapse cannot be simulated
properly
in vitro to date. Nevertheless, a non-inflamed cellular barrier lacking
attracting stimuli on
the basolateral side most likely reflects the conditions of basic immune
surveillance
which we consider as more important in terms of controlling a JCV reactivation
event.
Furthermore, the in vitro paradigms were designed to identify individuals at
risk of PML
on a large scale and therefore were kept as basic as possible to enable
maximum
experimental reproducibility.
[0330] Five patients in the inventors' cohort developed PML. One of these
patients has previously been described in a case report, mainly focusing on
the immune
response during PML and subsequent IRIS (Schwab, N., et al., Mult Scler.
(2012) 18,
335-344). Strikingly, all 5 PML patients shared three remarkable differences
to the rest
of the investigated cohort: 1) reduced transendothelial migration over the
complete time
frame, 2) missing LFA-1 recovery after 12 months, 3) missing CD62L expression
and
recovery. Data from Natalizumab-associated PML patients after plasma exchange
revealed that migration rates normalized after stopping the Natalizumab
treatment, while
CD62L expression only recovered to some extent. This might hint towards a
possible
pre-existing condition in some patients, possibly associated with a
predisposed shift in
effector/memory T-cell compartments (Schwab et al., 2012, supra). All patient
samples
at the beginning of the PML showed the very characteristic absence of CD62L
while
leaving the effector-memory percentages intact (assessed by CCR7). It should
be noted
that especially naive (CD45RA 'CCR7') CD4 T cells lacking the expression of
CD62L
do not exist in controls. CD62L might therefore be the first dynamic biomarker
linking
all different types of PML (antibody-associated concerning treatment with
Natalizumab,
Efalizumab, and Rituximab, as well as HIV-associated). Further studies need to
be
conducted to find out if the loss of CD62L contributes to the development of
PML or
whether it is not functionally associated, but rather symptomatic.
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[0331] Taken together, the above data support the assumption that part of the
clinical efficacy of Natalizumab is due to a selective inhibition of the T-
cell trafficking
route through the choroid plexus into the CNS responsible for the entry of
effector cells
during inflammatory events i.e. MS relapse. Absent recovery of
transendothelial
migration could result in impaired basic CNS immune surveillance, thereby
increasing
the risk for PML development. It cannot be excluded that other biomarkers
might also be
important in patients at enhanced risk for PML. Therefore, the inventors'
hypothesis
ought to be evaluated and expanded in larger cohorts. However, the inventors
would
suggest testing patients under long-term treatment for their capacity for
transendothelial
migration, their peripheral levels of LFA-1, and especially CD62L to assess
basic
immune competence. Patients showing compromised immune surveillance should be
clinically monitored very closely.
